Gentamycin Resistance Research Articles

Antimicrobial susceptibility and resistance mechanisms of methicillin resistant Staphylococcus aureus isolated from 12 Hospitals in Turkey.

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most important nosocomial pathogens and is also emerging in Turkish hospitals. The aim of this study was to determine the antimicrobial susceptibility profiles of MRSA isolated from Turkish hospitals.Materials and methodsA total of 397 MRSA strains isolated from 12 hospitals in Turkey were included to present study. Antimicrobial susceptibilities were tested using agar dilution method. Presence of ermA, ermB, ermC, msrA, tetM, tetK, linA and aac-aph genes were studied by PCR.ResultsAll strains were susceptible to vancomycin and linezolid. The susceptibility rates for fusidic acid, lincomycin, erythromycin, tetracyclin, gentamycin, kanamycin, and, ciprofloxacin were 91.9%, 41.1%, 27.2%, 11.8%, 8.5%, 8.3% and 6.8%, respectively. Lincomycin inactivation was positive for 3 isolates. Of 225 erythromycin resistant isolates 48 had ermA, 20 had ermC, and 128 had ermA-C. PCR was negative for 15 strains. Of 3 isolates with lincomycin inactivation one had linA and msrA. Of 358 gentamycin resistant isolates 334 had aac-aph and 24 were negatives. Among 350 tetracyclin resistant isolates 314 had tetM. Of 36 tetM negative isolates 10 had tetK.ConclusionMRSA isolates from Turkish hospitals were multiresistant to antimicrobials. Quinolone and gentamycin resistance levels were high and macrolide and lincosamide resistance were relatively low. Susceptibility rates for fusidic asid were high. Linezolide and vancomycin resistance are not emerged. The most common resistance genes were ermA, tetM and aac-aph. Evolution of antimicrobial susceptibilities and resistance genes profiles of MRSA isolates should be surveyed at regional and national level for accurate treatment of patients and to control dissemination of resistance genes.

Intestinal epithelial endocytosis of commensal bacteria is dependent on IFNγ‐induced terminal web myosin phosphorylation and brush border fanning

Bacterial dissemination is commonly seen in intestinal obstruction (IO). Paracellular influx was mediated by ROCK signaling for phosphorylated myosin light chain (pMLC) and tight junctional disruption. Myosin phosphorylation in terminal web (TW) is associated with brush border (BB) fanning. The mechanism of bacterial endocytosis in enterocytes remains unclear.AimTo investigate the role of IFNγ in TW pMLC for bacterial endocytosis.MethodsMouse intestines were obstructed by loop ligation in which inhibitors were luminally administered. Next, mice were injected i.p. with anti‐IFNγ. Bacterial endocytosis in isolated enterocytes was measured by gentamycin resistance assay and fluorescence in situ hybridization. Epithelial Ca2+/ATP‐dependent TW contraction and BB fanning were visualized using TEM.ResultsBacterial endocytosis was observed without paracellular permeability change at 3–6 h of IO. Enterocytic pMLC localized to TW was associated with BB fanning, which may be inhibited by ML‐7 but not Y27632. IO‐induced bacterial endocytosis through intermicrovillus space was reduced by ML‐7 or anti‐IFNγ. Caco‐2 cells treated with IFNγ showed MLCK‐dependent TW contraction and E. coli endocytosis and translocation.ConclusionsIFNγ‐induced epithelial MLCK‐dependent TW contraction and BB fanning is involved in bacterial endocytosis. Funding support by National Science Council and NTU.

Antimicrobial resistance in commensal Escherichia coli isolated from animals at slaughter

Monitoring of antimicrobial resistance in commensal Escherichia coli (N = 3430) isolated from slaughtered broilers, laying hens, turkeys, swine, and cattle in Poland has been run between 2009 and 2012. Based on minimal inhibitory concentration (MIC) microbiological resistance to each of 14 tested antimicrobials was found reaching the highest values for tetracycline (43.3%), ampicillin (42.3%), and ciprofloxacin (39.0%) whereas the lowest for colistin (0.9%), cephalosporins (3.6 ÷ 3.8%), and florfenicol (3.8%). The highest prevalence of resistance was noted in broiler and turkey isolates, whereas it was rare in cattle. That finding along with resistance patterns specific to isolation source might reflect antimicrobial consumption, usage preferences or management practices in specific animals. Regression analysis has identified changes in prevalence of microbiological resistance and shifts of MIC values. Critically important fluoroquinolone resistance was worrisome in poultry isolates, but did not change over the study period. The difference (4.7%) between resistance to ciprofloxacin and nalidixic acid indicated the scale of plasmid-mediated quinolone resistance. Cephalosporin resistance were found in less than 3.8% of the isolates but an increasing trends were observed in poultry and MIC shift in the ones from cattle. Gentamycin resistance was also increasing in E. coli of turkey and cattle origin although prevalence of streptomycin resistance in laying hens decreased considerably. Simultaneously, decreasing MIC for phenicols observed in cattle and layers isolates as well as tetracycline values in E. coli from laying hens prove that antimicrobial resistance is multivariable phenomenon not only directly related to antimicrobial usage. Further studies should elucidate the scope of commensal E. coli as reservoirs of resistance genes, their spread and possible threats for human and animal health.

Culture and Antibiotic Sensitivity of Escherichia coli Isolated from Patients with Urinary Tract Infections (UTI) in Jessore City

Urinary tract infection is caused by the presence and growth of microorganisms in the urinary tract, is perhaps the single commonest bacterial infection of mankind. The present study was carried out to identify Escherichia coli from urinary tract infected patients those come to a community hospital in Jessore city. For the confirmation of the presence of this disease causing agent various cultural and biochemical techniques were performed. A total of 87 clinically suspected patients having urinary tract infection were studied, 50 were found to be culture positive. Among the positive cultures, 30 (60%) isolates were infected with Escherichia coli. The study showed that the most susceptible group for urinary tract infection ranges between 16-45 years of age, as this group comprised of 15(50%) individuals. Antibiotic susceptibility for all 30 isolates was determined against locally available eleven drugs. High resistance rates were observed against most of the antibiotics examined here. Amoxycilin stood to be the most resistant type of antibiotic (90%), followed by nalidixic acid (57%), cephlexin (50%), ciprofloxacin (37%), ceftriaxone (27%), doxycycline (24%) and cefixime (20%). Gentamycin and nitrofurantoin got lower resistance rates at 14% and 3% respectively, but resistance to netilmycine was not found among all 30 isolates. Multidrug resistance pattern of the isolated organism was also observed in this present study.

Detection of High Level Aminoglycosides Resistant Entrococci In A Tertiary Care Hospital.

BACKGROUND: Emergence of high-level aminoglycoside and glycopeptide resistance has significantly contributed to the mortality, particularly in serious enterococcal infections. High Level Aminoglycoside Resistance (HLAR) is related to the slow uptake or permeability of these agents. AIM: The present study was undertaken to determine HLAR pattern of enterococci in our hospital. METERIALS AND METHODS: This study was done in the Department of Microbiology, Meenakshi medical college, during the period of February 2012- February 2013. A total of 52 enterococcal isolates were collected from various clinical samples and speciation was by a series of biochemical reactions as per standard protocol.. Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method and microbroth dilution method according to CLSI guidelines 2012. RESULT: In the present study majority of the Enterococcal isolates in our study were isolated from urine sample. Among the 52 isolates of enterococci E.faecalis was the predominant species. The highest percentage of antibiotic resistance was seen in Erythromycin followed by Ciprofloxacin, Tetracycline, Ampicillin and Vancomycin. The High level Gentamycin Resistance (HLGR) was 48.7% in E.faecalis and 54.6% in E.faecium & High Level Streptomycin Resistance (HLSR) was 34.1% in E.faecalis and 54.6% in E.faecium. 23 strains (17 E.faecalis and 6 E.faecium) showed resistance to High Level Gentamycin (HLG) in the concentration range of >500μg/ml. 17strains (12E.faecalis and 5 E.faecium) showed resistance to High Level streptomycin (HLS) at the range of >1000 μg/ml. CONCLUSION: This study emphasizes the need to screen for HLAR in patients suffering from enterococcal infections. Routine screening for high level aminoglycoside

Patterns of isolates in blood cultures and antibiotic resistance profiles over five years, in a tertiary surgical neonatal unit

BackgroundNeonatal sepsis with bacteraemia causes high morbidity and mortality. Early empirical antibiotics are essential to reduce this. Antibiotic policies are based on local epidemiological data of pathogens and resistance profiles.ObjectiveIdentify...

Antimicrobial resistance of Escherichia coli O 157:H7/NM isolated from feaces of ruminant animals in Iran

Bacterial resistance to antimicrobial agents is a phenomenon problem, and the understanding of resistance acquisition and transmission can contribute to the development of new strategies to the clinical and socio-economic implication of this phenomenon, especially in ruminant animals. A total of 327 fecal samples of ruminant animals were examined for E. coli O157:H7/NM and the antibiotic susceptibility testing was determined by disc diffusion according to the Clinical Laboratory Institute. Isolates were tested for susceptibility to antimicrobial drugs by the Kirby-Bauer disc diffusion method using MI‹ellar-Hinton agar according to the Clinical Laboratory Standards Institute. Twenty-five (7.6%)Escherichia coli O157:H7/NH were isolated, 24(96%) were resistant to one or more antibiotic agent. Six (24%) E. coli were resistant to one antibiotic agent, 11(44%) to two agents and 28% exhibited multi drug resistance. Gentamycin resistance phenotype was the most common (56.0%), followed by ampicillin (48.0%), erythromycin (40.0%), amoxicillin (16.0%), tetracycline (12.0%), chloramphenicol (8.0%) nalidixic acid (8.0%), and streptomycin (4.0%) and all E. coli O157 isolates were susceptible to cefuroxime. Considering the clinical implication of E. coli in veterinary medicine, surveillance of the bacterial pathogens became imperative for better understanding and infection control approach. Key words: Escherichia coli O 157, ruminant feces, antibiotic resistance, Iran.

Cell-to-cell movement of plastids in plants

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Normal conjunctival flora and their antibiotic sensitivity in Omanis undergoing cataract surgery

Purpose:To evaluate the normal conjunctival flora and to ascertain their sensitivity to antibiotics.Materials and Methods:Prospective study was undertaken with 56 patients scheduled for cataract surgery. In each of these patients, conjunctival swab was taken from both eyes before instilling any antibiotic drops or local anesthetics and were sent for culture sensitivity by inoculating the samples in blood agar plates. The un-operated eyes served as controls for the study. The results were tabulated and analyzed by applying confidence intervals at 95% confidence limits.Results:Of the 112 eyes studied, positive cultures were obtained from 54 eyes (48.3%) preoperatively, and included both operated and control eyes. Coagulase-negative staphylococcus was isolated in 44 eyes (81.5%). Maximum sensitivity was found with vancomycin, gentamycin, chloramphenicol, and ciprofloxacin and maximum resistance with fusidic acid, penicillin, and oxacillin.Conclusion:We conclude that coagulase-negative staphylococcus are the most common bacteria isolated from the conjunctival sac, and vancomycin followed by gentamycin and ciprofloxacin are most effective against normal conjunctiva l flora, and fusidic acid the most ineffective of all the antibiotics tested.

English

The aim of this study was to determine the antibiotic susceptibility pattern of Pseudomonas aeruginosa from sputum samples of lower respiratory tract infection (LRTI) patients admitted to the Department of Pediatrics and Pulmonary Medicine and also to update clinicians on various antimicrobial alternatives available in treatments. Sputum samples from 298 patients were cultured identified and antibiotic sensitivity was performed by standard methods. Out of the 298 sputum samples from LRTI patients 102 (34.23%) patients had established P. aeruginosa aetiology. Cefepime and ceftazidime show more resistance to P. aeruginosa isolates with resistance 36.27 and 35.30%, respectively. Least resistance was noted for piperacillin/tazobactam and Imipenam that is, 4.90 and 5.88%, respectively. Fifty percent P. aeruginosa are intermediate or marginally sensitive to gentamycin. Piperacillin/Tazobactam and Imipenam was found to be the most sensitive drug against P. aeruginosa in LRTI patients. Gentamycin resistance is continuously increasing.   Key words: Antibiotic susceptibility profile of P. aeruginosa, lower respiratory tract infection, multiple antibiotic resistance.

Investigation of antimicrobial susceptibility for enterococci isolated from cats and dogs and the determination of resistance genes by polymerase chain reaction

The aim of this study was to investigate the antimicrobial susceptibility and to determinate the resistance genes by Polymerase Chain Reaction (PCR) among enterococci isolated from healthy cats and dogs in İzmir, Turkey. From a total of 114 rectal swabs, 91 (31 cats and 60 dogs) stains were isolated as enterococci. Twenty eight isolates were identified as Enterococcus faecalis and 63 as Enterococcus faecium with the API 20 IDStrep system. Antimicrobial susceptibilities were determined by disc diffusion. Susceptibility rates of all enterococci strains were found as 100.0%, 95.6%, 94.5%, 87.9%, 84.6%, 30.8%, 29.7%, and 0.0% for teicoplanin, penicillin, vancomycine, chloramphenicol, gentamycin, erythromycin, tetracycline, and clindamicin, respectively. Of 91 enteroccocci isolates 14 (15.4%) were found susceptible to all antimicrobials tested while 77 (84.6%) strains were resistant. All isolates were found resistant to clindamicin while 5 E. faecium strains had an intermediate resistance to vancomycin. None of the intermediate resistant vancomycin strains has any vanA, vanB, or vanC genes. From 91 isolates 4, 11, 14, 63, and 64 were found resistant to penicillin, chloramphenicol, gentamycin, erythromycin, and tetracycline, respectively. Sixty two tetracycline resistant strains had tet(M) gene. From erythromycin resistant strains 2 and 54 had erm(A) and erm(B). Gentamycin resistance was detected for 14 isolates from which 10 had aac(6')-aph(2') genes. Seven strains have the cat gene of 11 chloramphenicol resistant strains. The results revealed that there was a high level of antimicrobial resistance among enterococci isolated from healthy cats and dogs and they pose a potential risk for transmission of enterococci to humans.

Phage typing, PCR amplification for mecA gene, and antibiotic resistance patterns as epidemiologic markers in nosocomial outbreaks of methicillin resistant Staphylococcus aureus

Staphylococcus aureus is one of the major causes of community and hospital-acquired infections. Bacteriophage considered as a major risk factor acquires S. aureus new virulence genetic elements. A total number of 119 S. aureus isolated from different specimens obtained from (RKH) were distinguished by susceptibility to 19 antimicrobial agents, phage typing, and PCR amplification for mecA gene. All of MRSA isolates harbored mecA gene, except three unique isolates. The predominant phage group is belonging to the (mixed group). Phage group (II) considered as an epidemiological marker correlated to β-lactamase hyper producer isolates. MRSA isolates indicated high prevalence of phage group (II) with highly increase for phage types (Ø3A), which were correlated to the skin. Phage types (Ø80/Ø81) played an important roll in Community Acquired Methicillin Resistant S. aureus (CAMRSA). Three outpatients MRSA isolates had low multiresistance against Bacitracin (Ba) and Fusidic acid (FD), considered as CAMRSA isolates. It was detected that group I typed all FD-resistant MSSA isolates. Phage groups (M) and (II) were found almost to be integrated for Gentamycin (GN) resistance especially phage type (Ø95) which relatively increased up to 20% in MRSA. Tetracycline (TE) resistant isolates typed by groups (II) and (III) in MSSA. Only one isolate resistant to Sulphamethoxazole/Trimethoprim (SXT) was typed by (III/V) alone in MSSA. MRSA isolates resistant to Chloramphenicol (C) and Ba were typed by all groups except (V). It could be concluded that (PERSA) S. aureus isolates from the wound that originated and colonized, and started to build up multi-resistance against the topical treatment antibiotics. In this study, some unique sporadic isolates for both MRSA and MSSA could be used as biological, molecular and epidemiological markers such as prospective tools.

Genetic Toggling of Alkaline Phosphatase Folding Reveals Signal Peptides for All Major Modes of Transport across the Inner Membrane of Bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.

Classification of plasmid vectors using replication origin, selection marker and promoter as criteria

Although plasmid DNA vectors have been extensively applied in biotechnology, there is still a lack of standard plasmid vector classification. Here, we propose a classification method for commonly used plasmid vectors. Plasmid vectors were classified into different classes based on their replication origin, selection marker and promoter information. The replication origins of plasmid vectors were classified as: prokaryotic replication origin, eukaryotic replication origin and viral replication origin. Selection markers of plasmid vectors were mainly classified as ampicillin, kanamycin, neomycin, chloramphenicol, gentamycin, tetracycline, erythromycin, streptomycin, vancomycin and spectinomycin resistance gene markers. Promoter sequences were also classified as prokaryotic, eukaryotic and viral promoters. Finally, the nomenclature of common plasmid vectors has three determinants. We believe that the classification of plasmid vectors can provide useful information for researchers employing molecular cloning procedures. A web service of the plasmid classification was established and it is available from http://www.computationalmedicalbiology.org/plasclas.aspx.

Species distribution and antimicrobial resistance of enterococci isolated from surface and ocean water

The species identification and antimicrobial resistance profiles were determined for enterococci isolated from Southern California surface and ocean waters. Species identification was determined for 1413 presumptive Enterococcus isolates from urban runoff, bay, ocean and sewage water samples. The most frequently isolated species were Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. All five of these species were isolated from ocean and bay water with a frequency ranging from 7% to 36%. Enterococcus casseliflavus was the most frequently isolated species in urban runoff making up 36-65% of isolates while E. faecium was the most frequently isolated species in sewage making up 53-78% of isolates. The similar distribution of species in urban runoff and receiving water suggests that urban runoff may be the source of Enterococcus. No vancomycin or high level gentamycin resistance was detected in E. faecalis and E. faecium isolates. Enterococcus faecalis, E. faecium, E. casseliflavus and E. mundtii are the most commonly isolated Enterococcus species from urban runoff and receiving waters in Southern California. Determination of the Enterococcus species isolated from receiving waters and potential pollution sources may assist in determining the sources of pollution.

High-Level Oxacillin and Gentamycin Resistance with Reduced Susceptibility to Vancomycin in Staphylococcus aureus-Carrying mecA and femA Gene Complex

Staphylococcus aureus oxiva 10 and oxiva 14 strains clinically isolated from diabetic patients were resistant to gentamycin and oxacillin The minimal inhibitory concentrations (MICs) of oxacillin and gentamycin were 720 and >2048 microg/mL, respectively, for oxiva 10 and 680 and 400 microg/mL. respectively, for oxiva 14; both strains carry mecA and femA genetic determinants in their genomes. In addition, both are vancomycin-intermediate Staphylococcus aureus (VISA) isolates. The addition of vancomycin led to significant decreases in oxacillin resistance of both oxiva 10 and oxiva 14 strains, whereas the addition of vancomycin to gentamycin plates showed a decrease in gentamycin resistance of non-high-level gentamycin-resistant (non-HLGR) oxiva 14 and indifference in gentamycin resistance in HLGR oxiva 10. Transmission electron microscopy of representative strains unveils a remarkable increase in the thickness of the cell wall, indicating that thickening of the cell wall is a common phenotype associated with vancomycin resistance in VISA isolates. The present study reports that the rate of synergism and synergistic effect in the combination vancomycin-gentamycin vary according to the MICs of gentamycin.

Correlation Between Antifungal Agent Phenazine-1-Carboxylic Acid and Pyoluteorin Biosynthesis in Pseudomonas sp. M18

Biosynthesis and secretion of two different types of antifungal compound [phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in Pseudomonas sp. M18] contribute to its suppression of soil-borne root pathogens. To better understand the correlation between two antifungal agents in secondary metabolism, a DNA fragment covering partial pltC and pltD coding sequences was obtained by screening the genomic library of Pseudomonas sp. M18. A mutant, M18T, was then constructed by insertion of the aacC1 gene cassette (encoding gentamycin resistance). With the same methods, one PCA biosynthetic gene cluster was insertionally inactivated and a mutant M18Z1 was created. The mutant strain M18T produces no Plt and the same amount of PCA in comparison with the wild-type strain M18. The mutant M18Z1, however, produces less PCA but more Plt than the wild-type strain M18. According to the documented data on strain M18, it is suggested that production of PCA is not influenced by Plt yield, but Plt biosynthesis is influenced by an alteration of PCA production.

Autoinduction of RpoS Biosynthesis in the Biocontrol Strain Pseudomonas sp. M18

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, sigma(S), or sigma(38)) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS'-'lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that beta-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its beta-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of beta-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS-lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.

Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta.

Bioaugmentation of Activated Sludge with Two Pseudomonas putida Strains for the Degradation of 4-Chlorophenol

The augmentation of activated sludge with two strains of Pseudomonas putida, CP1 and A(a) was investigated. Both strains of bacteria degraded 1.56 mM 4-chlorophenol. P. putida CP1 degraded the chemical using a modified ortho-cleavage pathway while P. putida A(a) used the meta-cleavage pathway. When activated sludge incapable of 4-chlorophenol degradation was augmented with either strain, substrate degradation occurred and followed the same biochemical pathways as when the bacteria were grown in pure culture. Insertion, in tandem, of the genes for gentamycin resistance and green fluorescent protein into the chromosomes of the two strains, enabled the survival and spatial location of the bacteria in the mixed microbial population to be monitored. Labelling the bacteria did not alter their degradative capabilities. P. putida CP1::Tn7-gfp survived in higher numbers than P. putida A(a)::Tn7-gfp following addition to the activated sludge. This was attributed to the ability of this strain to flocculate and become integrated in the activated sludge floc. Addition of P. putida CP1::Tn7-gfp or A(a)::Tn7-gfp to activated sludge resulted in smaller decreases in total cell numbers indicating a protective effect of the introduced P. putida strains on the overall microbial population from the harmful effects of 4-chlorophenol. The non-flocculant strain A(a) did not survive as well as CP1 in the activated sludge system and required a higher inoculum size to effect substrate degradation.