Occurrence Of Plasmids Research Articles

Occurrence and characterisation of ESBL-encoding plasmids among Escherichia coli isolates from fresh vegetables

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates have been increasingly reported in different reservoirs. The aims of this study were to investigate the presence of ESBL-producing E. coli in fresh vegetables and to characterise their ESBL gene-carrying plasmids. Among the 245 samples from vegetables investigated during 2011–2013, seven putative ESBL-producing E. coli (salad n = 2, sprouts n = 5) were found. They were subjected to ESBL phenotypic confirmatory tests, detection/sequencing of ESBL genes, antimicrobial susceptibility testing (AST), phylotyping, XbaI-macrorestriction analysis, multilocus sequence typing and transformation. Transformants were characterised by AST, S1-nuclease PFGE, replicon typing, conjugation and investigated for co-located antimicrobial resistance genes. Two ESBL gene-carrying plasmids were sequenced using a HiSeq 2500 system. The seven isolates were confirmed as ESBL producers, displayed unrelated XbaI-patterns and unique sequence types (STs) and belonged to the phylogroups A, B1 or D. The ESBL genes were located on plasmids. Two plasmids carrying blaCTX-M-14 genes (incompatibility group IncK or IncHI2) were seen in isolates from salad (ST973) and sprout (ST527). Two blaCTX-M-15- (IncFIB; non-typeable) and the IncN blaCTX-M-65- and IncHI2 blaCTX-M-125-carrying plasmids were found in isolates from sprouts (ST410, ST847, ST10, ST542). All plasmids were conjugative, except for the IncFIA-FIB blaCTX-M-2-carrying plasmid. Sequence analysis of two plasmids identified the ESBL genes in close location to other resistance genes: sulfonamide resistance gene sul2, streptomycin resistance genes strA and strB, the plasmid-mediated quinolone resistance gene qnrS1 and blaTEM-1 (sul2-strA-strB-IS66-blaTEM-1-tnpR-ΔtnpA-ISEcp1-blaCTX-M-15-Δorf477-ΔtnpA-qnrS1) or the fosfomycin resistance gene fosA3 (ΔISEcp1-blaCTX-M-125-ΔIS903B-fosA3). These observations underline the importance of vegetables as reservoirs for multidrug resistant ESBL-producing E. coli.

Occurrence of Plasmid- and Chromosome-Carried mcr-1 in Waterborne Enterobacteriaceae in China.

The aim of this study was to investigate the prevalence of the polymyxin resistance gene mcr-1 in Enterobacteriaceae from environmental water sources in Hangzhou, China. Colistin-resistant bacteria were isolated from environmental water samples using an enrichment broth culture method, were screened for mcr-1, and then were analyzed for the location and transferability of mcr-1 Isolates positive for mcr-1 were further examined to determine their susceptibility profiles and were screened for the presence of additional resistance genes. Twenty-three mcr-1-positive isolates (16 Escherichia coli, two Citrobacter freundii, two Klebsiella oxytoca, two Citrobacter braakii, and one Enterobacter cloacae) were isolated from 7/9 sampling locations; of those, eight mcr-1-positive isolates also contained β-lactamase-resistance genes, eight contained qnrS, and 10 contained oqx No mcr-2-positive isolates were identified. The majority of isolates demonstrated a low to moderate level of colistin resistance. Transconjugation was successfully conducted from 14 of the 23 mcr-1-positive isolates, and mcr-1 was identified on plasmids ranging from 60 to 220 kb in these isolates. Conjugation and hybridization experiments revealed that mcr-1 was chromosome-borne in only three isolates. Pulsed-field gel electrophoresis showed that the majority of E. coli isolates belonged to different clonal lineages. Multilocus sequence typing analysis revealed that sequence type 10 (ST10) was the most prevalent, followed by ST181 and ST206. This study demonstrates the utility of enrichment broth culture for identifying environmental mcr-1-positive isolates. Furthermore, it highlights the importance of responsible agriculture and clinical use of polymyxins to prevent further widespread dissemination of polymyxin-resistant pathogens.

Occurrence of plasmids in multi metal and drug resistant gram negative bacteria isolated From hospital wastewater

Occurrence of plasmids in multi metal and drug resistant gram negative bacteria isolated From hospital wastewater

Characterization of Arcobacter butzleri Cryptic Plasmid pDWAN from Human Isolate

Arcobacter butzleri is an underestimated bacterial pathogen cause of human gastroenteritis. This pathogen has been unexplored in terms of occurrence and depiction of plasmid. The sequence analysis reveals that one of the human isolate of A. butzleri contains cryptic plasmid pDWAN circular molecule of 1921 bp and G + C content of 27.5 %. There are two open reading frames present in pDWAN which is similar to RepB protein. One ORF is 100 % (105/105 aa) and another ORF is 98 % (174/177 aa) identical to RepB protein. Miscellaneous binding sites and unique restriction enzyme sites were identified within the plasmid. The plasmid pDWAN found that it maintained a stable genetic element. This is the first report showing occurrence of cryptic plasmid pDWAN in A. butzleri.

Plasmid Mediated Antibiotic and Heavy Metal Co-Resistance in Bacterial Isolates from Mahananda River Water (Malda, India)

Aims: The current investigation stands for the determination of MAR (multiple antibiotic resistances) indices of antibiotic resistant and heavy metal (HM) tolerant bacterial isolates from Mahananda river water near Malda (West Bengal state, India) and profiling of R-plasmids of the isolated bacteria. Methods: The water samples (n = 5), collected from Mahananda river water, Malda town, (India), were checked for the presence of bacterial growth in nutrient broth cultures. The pure bacteria colonies, procured from each of the nutrient broth cultures, were identified by conventional methods. The susceptibilities to antibiotics and HMs of the isolated bacteria were determined by disc diffusion and agar dilution, respectively. Plasmid curing of the resistant bacteria was done with SDS treatment, and the isolated plasmids were screened through agarose gel electrophoresis. The MAR indices for the isolates were calculated. Results: The isolated bacteria were identified as Escherichia coli (n = 3), and Pseudomonas aeruginosa (n = 2). The bacterial isolates showed various patterns of resistance to antibiotics and heavy metals. The MAR indices for the bacterial isolates ranged 0.0 – 0.2, for E. coli, and the value was 0.47, for both the Ps. aeruginosa isolates. The isolated bacteria harboured a single plasmid mediating co-resistance to antibiotics and HMs. Conclusion: The current study demonstrates the occurrence of plasmid, encoding Am-Cm-Ce-Tm-Cd2+-Hg2+ and Cm-Tm-Cx-Cd2+-Hg2+ resistances, among the aquatic bacteria of Mahananda river that may potentially act as the source of dissemination of pathogenic bacteria and bacterial antibiotic resistances, requiring public awareness on the issues of misuse and/or overuse of antibiotics.

Carbapenem-resistant Klebsiella pneumoniae isolates from Egypt containing blaNDM-1 on IncR plasmids and its association with rmtF.

The aim of this study was to characterize carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from clinical specimens at a tertiary care hospital in Egypt over a period of 15 months. Eight CRKP isolates were included in this study. The minimum inhibitory concentrations of different antibiotics were determined by broth microdilution and Etest methods. Multilocus sequence typing was performed. Antibiotic resistance genes were assessed by PCR and DNA sequencing. Plasmid analysis was done by S1 nuclease digestion of whole genomic DNA followed by pulsed-field gel electrophoresis (S1-PFGE). Eight carbapenem-resistant NDM-1-producing K. pneumoniae isolates of three different sequence types (ST) were identified (ST147, ST11, and ST17), in which blaNDM-1 was carried by either IncR or untypeable plasmids. Seven out of the eight isolates also contained the rmtF methylase gene. This study describes the occurrence of IncR plasmids carrying blaNDM-1 and rmtF in Egypt, raising concerns regarding this type of replicon and its role in the transmission of these resistance determinants.

Genomic and Functional Characterization of the Unusual pLOCK 0919 Plasmid Harboring the spaCBA Pili Cluster in Lactobacillus casei LOCK 0919.

Here, we report the extensive bioinformatic and functional analyses of the unusual pLOCK 0919, a plasmid originating from the probiotic Lactobacillus casei LOCK 0919 strain. This plasmid is atypical because it harbors the spaCBA-srtC gene cluster encoding SpaCBA pili. We show that all other spaCBA-srtC sequences of the Lactobacillus genus that have been previously described and deposited in GenBank are present in the chromosomal DNA. Another important observation for pLOCK 0919 is that the spaCBA-srtC gene cluster and its surrounding genes are highly similar to the respective DNA region that is present in the most well-known and active SpaCBA pili producer, the probiotic Lactobacillus rhamnosus GG strain. Our results demonstrate that the spaCBA-srtC clusters of pLOCK 0919 and L. rhamnosus GG are genealogically similar, located in DNA regions that are rich in transposase genes and are poorly conserved among the publicly available sequences of Lactobacillus sp. In contrast to chromosomally localized pilus gene clusters from L. casei and Lactobacillus paracasei, the plasmidic spaC of L. casei LOCK 0919 is expressed and undergoes a slight glucose-induced repression. Moreover, results of series of in vitro tests demonstrate that L. casei LOCK 0919 has an adhesion potential, which is largely determined by the presence of the pLOCK 0919 plasmid. In particular, the plasmid occurrence positively influenced the hydrophobicity and aggregation abilities of L. casei LOCK 0919. Moreover, in vivo studies indicate that among the three Lactobacillus strains used to colonize the gastrointestinal tract of germ-free mice, already after 2 days of colonization, L. casei LOCK 0919 became the dominant strain and persisted there for at least 48 days.

Susceptibility to ceftriaxone and occurrence of penicillinase plasmids in Neisseria gonorrhoeae strains isolated in Poland in 2012-2013.

Recent years have seen rising concerns over increasing antibiotic resistance of the gonorrhea-causing bacterium, Neisseria gonorrhoeae. This is especially true for third-generation cephalosporins, which are currently recommended for the treatment of such infections. Therefore, susceptibility to these antibiotics should be monitored internationally to the greatest extent possible. The susceptibility of N. gonorrhoeae strains to ceftriaxone and penicillin, as well as production of beta-lactamase by the Cefinase test was determined. Moreover, the presence and type of penicillinase plasmids were determined by PCR. All strains were susceptible to ceftriaxone, the minimal inhibitory concentration (MIC) values ranged from 0.002 to 0.125mg/L; MIC50 was =0.016mg/L and MIC90 was =0.064mg/L. As much as 7.7% of the strains demonstrated ceftriaxone MIC of 0.125mg/L. For penicillin, the MICs ranged from 0.064 to 32mg/L; MIC50 was =0.5mg/L and MIC90 was =4mg/L. It was shown that only 1.5% of the strains were sensitive to penicillin according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Among the penicillin-resistant strains, six (30.0%) produced penicillinase. The MICs of penicillin were substantially higher for penicillinase-producing than for penicillin-resistant, penicillinase-negative strains. MICs of ceftriaxone for penicillinase-producing strains were low (0.002-0.016mg/L). Three of the penicillinase-producing strains possessed plasmids of African type (50%) and three Toronto/Rio type (50%). An increase of the proportion of beta-lactamase-positive strains in the last years as well as emergence of strains with elevated MIC of ceftriaxone indicate a need to constantly monitor N. gonorrhoeae strains for their susceptibility to beta-lactam antibiotics, as well as for their ability to produce beta-lactamases.

Parallel Compensatory Evolution Stabilizes Plasmids across the Parasitism-Mutualism Continuum

Plasmids drive genomic diversity in bacteria via horizontal gene transfer [1, 2]; nevertheless, explaining their survival in bacterial populations is challenging [3]. Theory predicts that irrespective of their net fitness effects, plasmids should be lost: when parasitic (costs outweigh benefits), plasmids should decline due to purifying selection [4-6], yet under mutualism (benefits outweigh costs), selection favors the capture of beneficial accessory genes by the chromosome and loss of the costly plasmid backbone [4]. While compensatory evolution can enhance plasmid stability within populations [7-15], the propensity for this to occur across the parasitism-mutualism continuum is unknown. We experimentally evolved Pseudomonas fluorescens and its mercury resistance mega-plasmid, pQBR103 [16], across an environment-mediated parasitism-mutualism continuum. Compensatory evolution stabilized plasmids by rapidly ameliorating the cost of plasmid carriage in all environments. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Deletion of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Mutation of gacA/gacS downregulated the expression of ∼17% of chromosomal and plasmid genes and appears to have relieved the translational demand imposed by the plasmid. Chromosomal capture of mercury resistance accompanied by plasmid loss occurred throughout the experiment but very rarely invaded to high frequency, suggesting that rapid compensatory evolution can limit this process. Compensatory evolution can explain the widespread occurrence of plasmids and allows bacteria to retain horizontally acquired plasmids even in environments where their accessory genes are not immediately useful.

Resistance to Sulfonamides and Dissemination of sul Genes Among Salmonella spp. Isolated from Food in Poland.

Antimicrobial resistance of pathogenic bacteria, including Salmonella spp., is an emerging problem of food safety. Antimicrobial use can result in selection of resistant organisms. The food chain is considered a route of transmission of resistant pathogens to humans. In many European countries, sulfonamides are one of the most commonly used antimicrobials. The aim of our investigation was to assess the prevalence of sul genes and plasmid occurrence among sulfonamide-resistant Salmonella spp. Eighty-four sulfonamide-resistant isolates were collected in 2008 and 2013 from retail products in Poland. Minimal inhibitory concentration of all of these isolates was ≥1024 μg/mL. Resistant isolates were tested for the presence of sul1, sul2, sul3, and int1 genes by using multiplex polymerase chain reaction. In total, 44.0% (37/84) isolates carried the sul1 gene, 46.4% (39/84) were sul2 positive, while the sul3 gene was not detected in any of the sulfonamide-resistant isolates tested. It was found that 3.6% (3/84) of resistant Salmonella spp. contained sul1, sul2, and intI genes. All 33 intI-positive isolates carried the sul1 gene. Eleven of the sulfonamide-resistant isolates were negative for all the sul genes. Most of the sulfonamide-resistant Salmonella spp. harbored plasmids; only in eight isolates were no plasmids detected. Generally, the size of the plasmids ranged from approximately 2 kb to ≥90 kb. Our results revealed a relatively a high prevalence of sulfonamides-resistant Salmonella spp. isolated from retail food. Additionally, we have detected a high dissemination of plasmids and class 1 integrons that may enhance the spread of resistance genes in the food chain.

Plasmid Detection, Characterization, and Ecology.

Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. It is thought that to reduce the cost of plasmid carriage, only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness toward environmental changes. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance, and diversity of plasmids in environmental bacteria. Increasingly, cultivation-independent total-community DNA-based methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic-resistance-gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids, as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity, and evolution studies, but numerous challenges still exist.

IncP-1β Plasmids Are Important Carriers of Fitness Traits for Variovorax Species in the Mycosphere--Two Novel Plasmids, pHB44 and pBS64, with Differential Effects Unveiled.

The Laccaria proxima mycosphere strongly selects Variovorax paradoxus cells. Fifteen independent V. paradoxus strains, isolated from mycospheres sampled at two occasions, were investigated with respect to the occurrence of plasmids of sizes <60-100 kb. Two V. paradoxus strains, HB44 and BS64, were found to contain such plasmids, which were coined pHB44 and pBS64. Replicon typing using a suite of plasmid-specific PCR systems indicated that both plasmids belong to the IncP-1β group. Also, both were able to mobilize selectable IncQ group plasmids into Escherichia coli as well as Pseudomonas fluorescens. Moreover, they showed stable replication in these organisms, confirming their broad host range. Strain BS64 was cured of pBS64 and plasmid pHB44 was subsequently moved into this cured strain by making use of the IncQ group tracer plasmid pSUP104, which was then removed at elevated temperature. Thus, both plasmids could be screened for their ability to confer a phenotype upon strain BS64. No evidence for the presence of genes for xenobiotic degradation and/or antibiotic or heavy metal resistances was found for either of the two plasmids. Remarkably, both could stimulate the production of biofilm material by strain BS64. Also, the population densities of pBS64-containing strain BS64 were temporarily raised in liquid as well as soil systems (versus the plasmid-cured strain), both in the presence of the fungal host Lyophyllum sp. strain Karsten. Strikingly, plasmid pHB44 significantly enhanced the fitness of strain BS64 in soil containing Lyophyllum sp. strain Karsten, but decreased its fitness in soil supplemented with extra FeCl3. The effect was noted both in separate (no inter-strain competition) and joint (competition) inoculations.

Detection of multi drug resistant bacteria in major hospitals in Kano, North-West, Nigeria.

Two major hospitals in Kano, North West Nigeria have recorded increasing resistance of clinical pathogens to broad spectrum β lactams, mediated by extended spectrum β-lactamase (ESβL) and non ESBLs. A study was therefore undertaken to determine the occurrence and prevalence of plasmid and chromosomal mediated AmpC βL and carbapenemase in addition to already known ESBL due to increasing resistance of pathogens from the two hospitals to carbapenems, cephamycins and flouroquinolones. Antibiogram tests and ESBL, AmpC and carbapenemase production tests were performed on all the isolates. AmpC and carbapenemase producers were further screened for AmpC inducibility and metallo beta lactamase production respectively. Majority of the isolates (> 80%) were resistant to both β-lactam and non β-lactam antibiotics. Reduced susceptibility to levofloxacin, nitrofurantoin, nalidixic acid and ofloxacin among the isolates were observed with the exception of P. aeruginosa which is totally resistant to imipenem and levofloxacin. An overall prevalence of 14.4%, 11.9% and 11.9.3% for ESβL, AmpC and carbapenemase was observed respectively. About 7.9% of the AmpC producers can over expressed the chromosomally mediated AmpC and 85.8% of the carbapenemase producers require metal for their action. Co-production of either of two and/or all of the enzymes was observed in E. coli, P. mirabilis and P. aeruginosa. Antibiotic resistance among isolates from the two hospitals is increasing and the major cause of this resistance in the pathogens studied are production of AmpC, carbapenemase (especially Metallo β-lactamase) in addition to already known ESBL enzymes by the pathogens. Some of the isolates also possess the capacity to elaborate two or more of the enzymes concurrently, which would renders them resistant to a multitude of antibiotics.

Molecular characterization of extended spectrum β-lactamase (ESBL) producing Shigella species isolated from patients with bacillary dysentery in Iraq

Background: Extended-spectrum β-lactamases (ESBLs), including cefotaximases (CTX-M), mediate resistance to extended spectrum cephalosporins and significantly compromise the treatment tools for Shigellosis.&#x0D; Objective: To determine the ESBLs production by Shigella spp. and its role in the resistance to third generation cephalosporins and to determine the occurrence of plasmid- borne blaCTX genes in ESBLS Shigella isolates by Multiplex PCR.&#x0D; Methods: Susceptibility of 59 clinical Shigella isolates was tested by disk diffusion method to six antimicrobial agents. Presence of ESBLs was established by the combination disk method. Minimum inhibitory concentration of β-lactams was determined by agar dilution method for resistant isolates, and then ESBL producers were subjected to plasmid extraction and PCR experiments.&#x0D; Results: Overall isolates had high rate of resistance to Ampicillin (84.7%), Tetracycline (84.7%), Co-trimoxazole(72.9%) and Cefotaxime(69.5%). Moderate to Ceftriaxone (52.5%) and low rate of resistance to Ceftazidime(30.5%). The MIC value of resistant isolates of S. sonnei and S. flexneri for CTX and CRO ranged from (64-512 μg/ml) for both, and (32-256 μg/ml) for CAZ, whereas S. dysenteriae exhibit only high level resistance MIC (256 μg/ml) for CTX, low level resistance (64 μg/ml) for CRO and complete susceptibility to CAZ. 31 of 41 (75.6 %) of Shigella spp. were β-lactamase producers, of these (82.6%) were S. flexneri, (68.8%) S. sonnei, and one S. dysenteriae isolate. The results of PCR amplification of bla CTX gene groups showed that blaCTX-M genes were identified in (74.19%) ESBLs isolates distributed as 14(45.16%) S.flexneri isolates, 9(29.03%) S. sonnei, while no gene was detected in ESBLs S. dysenteriae isolate. The prevalence of bla CTX-M I, bla CTX-M II (TOHO 1) and blaCTX-M III, were ( 54.8%, 16.1% and 3.2% ) respectively. Non of the isolates carried more than one type.&#x0D; Conclusion: The occurrence of plasmid- borne blaCTX genes in ESBLS Shigella isolates are increasing in Iraq with co-resistance to some other classes of antibiotics

A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences

BackgroundIn vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. However, the current in vivo assembly methods are not consistent with respect to yields of correctly assembled constructs and standardization of parts required for routine laboratory implementation has not been explored. Here, we present and evaluate an optimized and robust method for in vivo assembly of plasmids from overlapping DNA fragments in S. cerevisiae.ResultsTo minimize occurrence of misassembled plasmids and increase the versatility of the assembly platform, two main improvements were introduced; i) the essential elements of the vector backbone (yeast episome and selection marker) were disconnected and ii) standardized 60 bp synthetic recombination sequences non-homologous with the yeast genome were introduced at each flank of the assembly fragments. These modifications led to a 100 fold decrease in false positive transformants originating from the backbone as compared to previous methods. Implementation of the 60 bp synthetic recombination sequences enabled high flexibility in the design of complex expression constructs and allowed for fast and easy construction of all assembly fragments by PCR. The functionality of the method was demonstrated by the assembly of a 21 kb plasmid out of nine overlapping fragments carrying six glycolytic genes with a correct assembly yield of 95%. The assembled plasmid was shown to be a high fidelity replica of the in silico design and all glycolytic genes carried by the plasmid were proven to be functional.ConclusionThe presented method delivers a substantial improvement for assembly of multi-fragment expression vectors in S. cerevisiae. Not only does it improve the efficiency of in vivo assembly, but it also offers a versatile platform for easy and rapid design and assembly of synthetic constructs. The presented method is therefore ideally suited for the construction of complex pathways and for high throughput strain construction programs for metabolic engineering purposes. In addition its robustness and ease of use facilitate the construction of any plasmid carrying two or more genes.

Comparative analysis and distribution of pP9014, a novel drug resistance IncP-1 plasmid from Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida, a causative agent of pseudotuberculosis, often harbours resistance plasmids (R plasmids) that facilitate horizontal gene transfer of drug resistance genes. R plasmid pP9014 was isolated from P. damselae subsp. piscicida and its complete nucleotide sequence was determined using Next Generation Sequencing technology. A protein network analysis was conducted to determine the relatedness of protein coding sequences, and ClustalW was used for the full nucleotide sequences. The occurrence of pP9014-like plasmids compared with pP99-018-like plasmids in a specific region was determined using probes for their transfer regions. pP9014 is 55851bp long with an overall GC content of 44.4% encoding 61 open reading frames (ORFs) including antimicrobial resistance genes and two conjugative transfer regions (Tra and Trb). The backbone showed highest similarity to Marinobacter adhaerens pHP-42 and Methylophaga sp. JAM7. pP9014 is similar to several IncP plasmids but forms a different subgroup. pP9014 is a unique plasmid in P. damselae subsp. piscicida and was not commonly found in drug-resistant P. damselae subsp. piscicida isolated from different areas and years in Japan. Plasmids similar to the previously reported pP99-018 are more widely distributed. This rarity suggests that plasmids similar to pP99-018 are more compatible with γ-proteobacteria. pP9014 is the first reported IncP-1 plasmid from fish pathogens. Its similarity to other IncP plasmids isolated from soil and human pathogens suggests that plasmids of the IncP-1 incompatibility group are vectors for the transfer of drug resistance genes among diverse environments.

Relationship between plasmid occurrence and antibiotic resistance in Myroides odoratimimus SKS05-GRD isolated from raw chicken meat

Fifteen flavobacterium strains were isolated from raw chicken meat, raw goat meat and poultry soil in Coimbatore, Tamil Nadu. Most of the isolates developed yellow pigmented colonies with mucoid-spreading edges on food flavobacterium medium. The flavobacteria were Gram-negative rods and failed to produce indole and were non-fermentative. Moreover, they produced a rich array of enzymes such as amylase, lipase, catalase, urease, gelatinase, DNase, and oxidase. Phylogenetic analyses of the strain SKS05-GRD based on 16S rRNA gene sequences revealed the bacterium as Myroides odoratimimus (nucleotide sequence accession number JQ178355). Antimicrobial susceptibility test for M. odoratimimus SKS05-GRD and other strains were assessed by disc diffusion method. M. odoratimimus SKS05-GRD showed wide resistance to the antibiotics such as amikacin, ampicillin, cefadroxil, cefoperazone, ceftazidine, ceftriaxone, netillin and gentamicin. M. odoratimimus was subjected to plasmid isolation and plasmid curing to seek the relationship between plasmid and antibiotic resistance. Plasmid curing was done by using ethidium bromide and was found to be effective at 300 and 500μg/ml. Assessment of antibiotic sensitivity of M. odoratimimus SKS05-GRD showed sensitivity to amikacin, gentamicin and kanamycin confirming that resistance to these three antibiotics is plasmid mediated and other antibiotic resistance are chromosomal mediated.

Plant Growth Promoting and Metal Bioadsorption Activity of Metal Tolerant Pseudomonas aeruginosa Isolate Characterized from Uranium Ore Deposit

In the present study, a metal-tolerant fluorescent pseudomonad isolated from uranium ore rich deposit of Domiasiat in North-East India was identified as Pseudomonas aeruginosa DPs-13 based on morphological, biochemical and molecular analysis. The isolate showed higher tolerance to uranium and other metals like copper, cadmium, zinc and lead when compared with the reference strain P. aeruginosa MTCC2474. When checked for uranyl bioadsorption potential, the isolate showed 94 % (22.5 mg/L) and 72 % (342.7 mg/L) removal of uranium (VI) when challenged with 100 μM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions within 1 h of incubation as compared to 68 % (16.18 mg/L) and 25 % (119 mg/L) when challenged with above concentrations respectively of uranyl nitrate by the reference strain. The isolate was resistant to most of the commonly used antibiotics like Ampicillin, Kanamycin, Chloramphenicol, Erythromycin, Aztreonam, Tetracycline, Ciprofloxin and Streptomycin. The isolate had no phytotoxic effect, produced siderophores, possessed phosphate solubilising ability as well as two antibiotic producing genes, and had antagonistic activity against plant pathogens. Plasmid occurrence was also noticed in the isolate. The isolate from the uranium ore rich site besides being a promising metal tolerant bacterium had potent plant growth promoting activity and can be used to promote plant growth in bioremediation approaches in metal contaminated sites.

Prevalence, antibiotic resistance and plasmid profiling of Salmonella in catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from wet markets and ponds in Malaysia

The objectives of the study were to determine the prevalence, antibiotic resistance and occurrence of plasmids in Salmonella isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from wet markets and ponds in Malaysia. A total of 172 samples (32 catfish carcass rinse, 32 catfish intestines, 32 tilapia carcass rinse, 32 tilapia intestines, and 44 water samples) were obtained from nine wet markets and eight ponds that were fed chicken offals, spoiled eggs, and commercial fish feed from 2008 to 2009. Seven Salmonella serovars were isolated from 9/32 catfish (28.1%), 14/32 tilapia (43.8%), and 11/44 (25%) water samples. These include S. Albany, S. Agona, S. Corvallis, S. Stanley, S. Typhimurium, S. Mikawashima and S. Bovis-mobificans. Salmonella isolates were resistant to chloramphenicol (C, 37.2%), clindamycin (Da, 100%), rifampicin (Rd, 90.7%), spectinomycin (Sh, 27.9%), and tetracycline (Te, 67.4%). The multiple antibiotic resistance index of Salmonella isolates ranged from 0.32 to 0.45 for catfish; 0.14 to 0.36 for tilapia; and 0.27 to 0.36 for water. The predominant antimicrobial resistance profiles of Salmonella serovars from catfish, tilapia and water were CDaRdTe (4/13), DaRdSh (4/19), and DaRdTe (6/11), respectively. The plasmids of Salmonella serovars isolated from catfish ranged from 23 to 80kb; those for tilapia ranged from 6 to 90kb; that for water ranged from 6 to 70kb, respectively. The presence of plasmids represents a potential health hazard since plasmids can mediate the transfer of antibiotic resistance genes to other bacteria present in the fish, and aquaculture environment, which can also enter the food chain.

High frequency of co-resistance in CTX-M-producing Escherichia coli to non-beta-lactam antibiotics, with the exceptions of amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin, in a county of Sweden

The objective of this study was to investigate the in vitro activity of different antibiotics against CTX-M-producing Escherichia coli in a county of Sweden, and to determine the occurrence of multi-resistance and plasmid- mediated quinolone resistance among these isolates. A total of 198 isolates of E. coli with extended-spectrum beta-lactamase (ESBL) phenotype and mainly CTX-M genotype were studied. The minimum inhibitory concentrations (MICs) for amikacin, chloramphenicol, ciprofloxacin, colistin, fosfomycin, gentamicin, nalidixic acid, nitrofurantoin, tigecycline, tobramycin, trimethoprim, and trimethoprim-sulfamethoxazole were determined with the Etest. Susceptibility was defined according to the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MIC50 and MIC90 values were calculated. Ninety-five percent or more of the isolates were susceptible to amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin. CTX-M group 9 was more susceptible than CTX-M group 1 to ciprofloxacin, gentamicin, and tobramycin. Sixty-eight percent of the isolates were multi-resistant, and the most common multi-resistance pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin, and tobramycin. Only 1 isolate carried a qnrS1 gene, but 37% carried aac(6')-Ib-cr. A high frequency of co-resistance between ESBL-producing E. coli and non-beta-lactam antibiotics was seen. On the other hand, very high susceptibility was seen for amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin. These data support the replacement of gentamicin and tobramycin, normally used in Sweden, with amikacin, for severe infections.