Plasmid-bearing Strains Research Articles

Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria.

The ability to genetically manipulate a pathogen is fundamental to discovering factors governing host-pathogen interactions at the molecular level and is critical for devising treatment and prevention strategies. While the genetic "toolbox" for many important bacterial pathogens is extensive, approaches for modifying obligate intracellular bacterial pathogens were classically limited due in part to the uniqueness of their obligatory lifestyles. Many researchers have confronted these challenges over the past two and a half decades leading to the development of multiple approaches to construct plasmid-bearing recombinant strains and chromosomal gene inactivation and deletion mutants, along with gene-silencing methods enabling the study of essential genes. This review will highlight seminal genetic achievements and recent developments (past 5 years) for Anaplasma spp., Rickettsia spp., Chlamydia spp., and Coxiella burnetii including progress being made for the still intractable Orientia tsutsugamushi. Alongside commentary of the strengths and weaknesses of the various approaches, future research directions will be discussed to include methods for C. burnetii that should have utility in the other obligate intracellular bacteria. Collectively, the future appears bright for unraveling the molecular pathogenic mechanisms of these significant pathogens.

Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain.

Xylella fastidiosa (Xf) is a phytopathogenic bacterium with a repertoire of self-replicating genetic elements, including plasmids, pathogenicity islands, and prophages. These elements provide potential avenues for horizontal gene transfer both within and between species and have the ability to confer new virulence traits, including the ability to colonize new host plants. However, they can also serve as a ‘footprint’ to type plasmid-bearing strains. Genome sequencing of several strains of Xf subsp. fastidiosa sequence type (ST) 1 from Mallorca Island, Spain, revealed the presence of a 38 kb plasmid (pXFAS_5235). In this study, we developed a PCR-based typing approach using primers targeting the traC gene to determine the presence of pXFAS_5235 plasmid or other plasmids carrying this gene in a world-wide collection of 65 strains X. fastidiosa from different subspecies and STs or in 226 plant samples naturally infected by the bacterium obtained from the different outbreaks of Xf in Spain. The traC gene was amplified only in the plant samples obtained from Mallorca Island infected by Xf subsp. fastidiosa ST1 and from all Spanish strains belonging to this ST. Maximum-likelihood phylogenetic tree of traC revealed a close relatedness among Spanish and Californian strains carrying similar plasmids. Our results confirm previous studies, which suggested that a single introduction event of Xf subsp. fastidiosa ST1 occurred in the Balearic Islands. Further studies on the presence and role of plasmids in Xf strains belonging to the same or different subspecies and STs can provide important information in studies of epidemiology, ecology, and evolution of this plant pathogen.

Use of traC gene to type the incidence and distribution of pXFAS_5235 plasmid-bearing strains of X. fastidiosa subsp. fastidiosa ST1 in Spain

Use of traC gene to type the incidence and distribution of pXFAS_5235 plasmid-bearing strains of X. fastidiosa subsp. fastidiosa ST1 in Spain

Molecular epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae in China

ABSTRACT The epidemiological features of the newly emerged carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and its potential threat to human health are currently unknown. In this study, a total of 784 bla KPC-2-bearing CRKP strains collected from three hospitals located at different geographical locales in China during 2014–2017 were subjected to molecular typing, screening of virulence plasmid, string test and WGS (367/784 strains). The proportion of CRKP among all clinical K. pneumoniae strains increased sharply in China during 2014–2017. A large proportion (58%) of these CRKP strains were found to harbour a virulence-encoding plasmid, while only 13% of such strains exhibited a hypervirulent phenotype by string test and neutrophil assay. The lack of hypervirulent phenotype in virulent plasmid-bearing CRKP strains was found to be due to the mutation’s presence on rmpA and rmpA2 genes, which rendered them non-functional, while some strains carrying wild type rmpA did not exhibit hypervirulent phenotype either suggesting that other factors might also contribute to the hypervirulence of CRKP. Phylogenetic and SNP analysis indicated that the transmission of these CRKP strains in China likely involved several major clones of ST11. Carriage of IncFII pSWU01-like, bla KPC-2-bearing plasmid was found to be the major mechanism of carbapenem resistance in these CRKP strains. In conclusion, our data indicated that the prevalence of CRKP strains carrying the virulence plasmid has rapidly increased in China, while genetic markers were not correlated well with the hypervirulent phenotypes, which call for a better definition and screening for these truly hypervirulent CR-HvKP strains in clinical settings.

Interspecies transfer of plasmid-borne gentamicin resistance between Staphylococcus isolated from domestic dogs to Staphylococcus aureus.

As preconized by the One Health concept, the intimate relationship between pets and owners is a common source for the trade of microorganisms with zoonotic potential, and with them, antimicrobial resistance genes. In this work, we evaluated the presence of antimicrobial resistance genes, that are usually within mobile genetic elements, in a laboratory collection of 79 canine Staphylococcus strains, mostly Staphylococcus pseudintermedius and Staphylococcus coagulans. Resistance to tetracycline was observed in 34% of the strains, followed by resistance to erythromycin (21%) and gentamicin (19%). These phenotypes were partially correlated with the presence of the tetracycline resistance genes tet(M) and tet(K) in 64% and 44% of all strains, respectively; erythromycin resistance genes erm(A) and erm(C) in 53% and 23%; and gentamicin resistance gene aac(6')-aph(2″) in 26% of the strains. At least 45% of the strains harbored high- and/or low-molecular weight plasmids, whose transfer may be facilitated by their widespread biofilm-forming capacity, and absence of restrictive CRISPR systems. We selected eight plasmid-bearing and multidrug resistant strains, which were submitted to plasmid curing by stress with SDS. No strain lost resistance during stressing cultivation but, by conjugation experiments, the S. pseudintermedius strain 27 transferred its plasmid-borne resistance to gentamicin, conferred by the aac(6')-aph(2″) gene, to Staphylococcus aureus. The frequent empirical use of gentamicin to treat skin and ear infections in domestic dogs is likely to select resistant strains. Also, as demonstrated by our study, these strains can serve as gene reservoirs for human pathogens, such as S. aureus.

Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana benthamiana.

Metabolic engineering of heterologous hosts requires coexpression of multiple genes, often more than ten for a single pathway. Traditional approaches to create genetic constructs for this purpose are highly inflexible and suffer from very low throughput. In this book chapter, we describe a powerful method to overcome this bottleneck, namely, combinatorial co-expression in the Australian tobacco plant Nicotiana benthamiana. This system is based on Agrobacterium tumefaciens-mediated transient gene expression, often called agroinfiltration. Herein, instead of creating complex multigenic constructs, coexpression is achieved by simply mixing different plasmid-bearing Agrobacterium strains without the need for different selection markers. We present a practical guide for coexpressing multiple biosynthetic genes followed by GC-MS analysis, using production of the plant triterpene β-amyrin as an example. Our chapter provides a guideline to harness the potential of this versatile expression system in the natural product community for studying and engineering metabolic pathways.

A Large-Scale Sequencing-Based Survey of Plasmids in Listeria monocytogenes Reveals Global Dissemination of Plasmids.

The food-borne pathogen Listeria monocytogenes is known for its capacity to cope with multiple stress conditions occurring in food and food production environments (FPEs). Plasmids can provide benefits to their host strains, and it is known that various Listeria strains contain plasmids. However, the current understanding of plasmid frequency and function in L. monocytogenes strains remains rather limited. To determine the presence of plasmids among L. monocytogenes strains and their potential contribution to stress survival, a comprehensive dataset was established based on 1,921 published genomes from strains representing 14 L. monocytogenes sequence types (STs). Our results show that an average of 54% of all L. monocytogenes strains in the dataset contained a putative plasmid. The presence of plasmids was highly variable between different STs. While some STs, such as ST1, ST2, and ST4, contained few plasmid-bearing strains (<15% of the strains per ST), other STs, such as ST121, ST5, ST8, ST3, and ST204, possessed a higher proportion of plasmid-bearing strains with plasmids found in >71% of the strains within each ST. Overall, the sizes of plasmids analyzed in this study ranged from 4 to 170 kbp with a median plasmid size of 61 kbp. We also identified two novel groups of putative Listeria plasmids based on the amino acid sequences of the plasmid replication protein, RepA. We show that highly conserved plasmids are shared among Listeria strains which have been isolated from around the world over the last few decades. To investigate the potential roles of plasmids, nine genes related to stress-response were selected for an assessment of their abundance and conservation among L. monocytogenes plasmids. The results demonstrated that these plasmid genes exhibited high sequence conservation but that their presence in plasmids was highly variable. Additionally, we identified a novel transposon, Tn7075, predicted to be involved in mercury-resistance. Here, we provide the largest plasmid survey of L. monocytogenes to date with a comprehensive examination of the distribution of plasmids among L. monocytogenes strains. Our results significantly increase our knowledge about the distribution, composition, and conservation of L. monocytogenes plasmids and suggest that plasmids are likely important for the survival of L. monocytogenes in food and FPEs.

Within-host bacterial growth dynamics with both mutation and horizontal gene transfer.

The evolution and emergence of antibiotic resistance is a major public health concern. The understanding of the within-host microbial dynamics combining mutational processes, horizontal gene transfer and resource consumption, is one of the keys to solving this problem. We analyze a generic model to rigorously describe interactions dynamics of four bacterial strains: one fully sensitive to the drug, one with mutational resistance only, one with plasmidic resistance only, and one with both resistances. By defining thresholds numbers (i.e.each strain's effective reproduction and each strain's transition threshold numbers), we first express conditions for the existence of non-trivial stationary states. We find that these thresholds mainly depend on bacteria quantitative traits such as nutrient consumption ability, growth conversion factor, death rate, mutation (forward or reverse), and segregational loss of plasmid probabilities (for plasmid-bearing strains). Next, concerning the order in the set of strain's effective reproduction thresholds numbers, we show that the qualitative dynamics of the model range from the extinction of all strains, coexistence of sensitive and mutational resistance strains, to the coexistence of all strains at equilibrium. Finally, we go through some applications of our general analysis depending on whether bacteria strains interact without or with drug action (either cytostatic or cytotoxic).

Formation of new PHE plasmids in pseudomonads in a phenol-polluted environment

Several years ago, a laboratory-constructed plasmid with a single-component phenol monooxygenase gene (pheBA operon) flanked by two IS elements was released to a phenol-polluted area. During the following years, we found in the test area widely distributed pheBA operon-containing bacteria. The new pheBA+ strains belong predominantly to the Pseudomonas fluorescens group, and they did not arise via selection of the released PHE plasmid. On the contrary, the formation of several different types of PHE plasmids occurred, namely pPHE101 (60,958 bp) from the IncP-9 group, non-transferable plasmid pPHE69 (44,717 bp), mobilizable plasmid pPHE20 (39,609 bp) and the IncP-7 type plasmid pPHE24ΔpheBA (120,754 bp), in which the pheBA operon was translocated from the plasmid to the chromosome. In two cases, PHE plasmid-bearing strains exist in a multi-plasmid state, also containing the non-catabolic plasmids pG20 (133,709 bp) and pG69 (144,433 bp) with backbones sharing 97% DNA identity and with redundant genes for the initiation of replication, repA1and repA2, of which only one was active. Seemingly, several other plasmids and bacterial features besides the pheBA operon were involved in selective distribution of catabolic operons in the natural environment. The comparison of the genetic structure of plasmids and IS elements' functions, as well as resistance to heavy metals of seven completely sequenced plasmids, are discussed.

Salmonella-Infected Aortic Aneurysm: Investigating Pathogenesis Using Salmonella Serotypes.

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes – Typhimurium, Enteritidis, and Choleraesuis – in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1β, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy – significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1β secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1β secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.

Hotspot mutations and ColE1 plasmids contribute to the fitness of Salmonella Heidelberg in poultry litter.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is a clinically-important serovar linked to food-borne illness, and commonly isolated from poultry. Investigations of a large, multistate outbreak in the USA in 2013 identified poultry litter (PL) as an important extra-intestinal environment that may have selected for specific S. Heidelberg strains. Poultry litter is a mixture of bedding materials and chicken excreta that contains chicken gastrointestinal (GI) bacteria, undigested feed, feathers, and other materials of chicken origin. In this study, we performed a series of controlled laboratory experiments which assessed the microevolution of two S. Heidelberg strains (SH-2813 and SH-116) in PL previously used to raise 3 flocks of broiler chickens. The strains are closely related at the chromosome level, differing from the reference genome by 109 and 89 single nucleotide polymorphisms/InDels, respectively. Whole genome sequencing was performed on 86 isolates recovered after 0, 1, 7 and 14 days of microevolution in PL. Only strains carrying an IncX1 (37kb), 2 ColE1 (4 and 6kb) and 1 ColpVC (2kb) plasmids survived more than 7 days in PL. Competition experiments showed that carriage of these plasmids was associated with increased fitness. This increased fitness was associated with an increased copy number of IncX1 and ColE1 plasmids. Further, all Col plasmid-bearing strains had hotspot mutations in 37 loci on the chromosome and in 3 loci on the IncX1 plasmid. Additionally, we observed a decrease in susceptibility to tobramycin, kanamycin, gentamicin, neomycin and fosfomycin for Col plasmid-bearing strains. Our study demonstrates how positive selection from poultry litter can change the evolutionary path of S. Heidelberg.

Complete Genome Sequences of the Plasmid-Bearing Campylobacter coli Strains HC2-48, CF2-75, and CO2-160 Isolated from Retail Beef Liver.

The complete genome sequences of Campylobacter coli strains HC2-48, CF2-75, and CO2-160, isolated from retail beef liver, showed the presence of 1,663,782-, 1,711,393-, and 1,683,224-bp circular chromosomes and 44,064-, 44,233-, and 44,228-bp circular plasmids, respectively. This is the first reported Campylobacter coli genome sequence isolated from retail beef liver.

Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids

In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

An enhanced biodegradation of crude oil by Psedomonas plasmid-bearing strains in model soil systems

Oil biodegradation in sterile and nonsterile model soil systems has been studied. A comparison of process efficiency using indigenous microorganisms, introduced plasmid-free bacteria, and strains bearing various plasmids of naphthalene degradation was carried out. It was shown, in almost all variants of the experiment, that the total oil reduction in the nonsterile soil was higher than in the sterile soil. The highest level of degradation was observed in soil systems containing plasmid-bearing strains.

Effect of the expression of Escherichia coli fhuA gene in Rhizobium sp. IC3123 and ST1 in planta: Its role in increased nodule occupancy and function in pigeon pea

The inability to utilize a fungal siderophore as source of iron nutrition by most of the rhizobial cultures isolated from pigeon pea, could be considered a negative fitness factor since hydroxamate siderophores are found in significant amounts in natural soils. Thus these cultures were engineered to use ferrichrome a prototype of hydroxamate type siderophore. Pigeon pea Rhizobium spp. IC3123 and ST1 harboring Escherichia coli fhuA gene, responsible for uptake of Fe 3+-ferrichrome, were obtained by transformation with pGR1, a broad host range plasmid carrying the fhuA gene under the control of the lac promoter of E. coli. Expression of fhuA in transformed rhizobial strains IC3123::pGR1 and ST1::pGR1 was confirmed by the ability of the plasmid-bearing strains to utilize iron bound to ferrichrome. Inoculation of pigeon pea plants with fhuA expressing rhizobial strains in pot experiments showed a significant increase in plant growth as well as nodule density as compared to those inoculated with the parent as well as the empty vector-bearing strain. Inoculation of pigeon pea seedlings with IC3123::pGR1 and ST1::pGR1 led to marked increase in shoot fresh weight, nodule number per plant, chlorophyll content of leaves and effective nodule symbiosis when compared with plants inoculated with the parent strains IC3123 and ST1. The positive effect of IC3123::pGR1 and ST1::pGR1 treatment on plant growth was more significantly observed when ferrichrome producing Ustilago maydis, known to secrete ferrichrome, was co-inoculated along with the transformed rhizobia. The presence of fhuA gene in rhizobial strains also led to an increased survival and root colonization.

Distribution Pattern and Physical Mapping of CCC Plasmid-Mediated Multidrug-Resistance in some G-ve Bacteria Recovered from Hospitals and Haemodialysis Units Wastewater at Al-Madinah Al-Munawarah

Abstract Objective Investigation of the presence and distribution pattern of plasmid-linked antibiotic resistance amongst Gram-negative bacterial strains recovered from wastewater of hospitals and haemodialysis units. Drawing up a physical map of the most frequently distributed plasmids Methods Representatives G-ve bacterial strains; 59 isolates, were chosen from a previous study for the same authors, as of the most resistant ones (resist more than 3 up to 9 antibiotics to > 100 μg/ml.). Miniprep protocol and gel electrophoresis technique were adopted for the detection and isolation of ccc plasmid DNA from the differentially-isolated G-ve bacteria. Restriction analysis using 11 DNA restriction enzymes and “Plasp” computer program against λ phage DNA digested with Hind III, for physical mapping of the most distributed plasmid. Results Results revealed the presence of seven different plasmids, distributed as 1-3 different types of plasmids in 68% (40/59) of the strains; chosen as representatives of the most resistant ones (resist more than 3 up to 9 antibiotics to > 100 μg/ml.). The detected plasmid sizes were estimated against HindIII-digested λ phage and found ranged from 36,844 to 2,027 bp. Preliminary physical mapping was constructed for the plasmid (23130 bp) that was the most distributed amongst more than 90% of the plasmid-bearing strains and was the only found singly. Conclusion The isolated, characterized and the mapped plasmid (s), suggest the possibility of high rate of both vertical and horizontal distribution of these plasmids amongst the isolated G-ve bacterial Genera and species.

Evolution of altruists and cheaters in near-isogenic populations of Escherichia coli

Emergence of antibiotic-resistant bacteria threatens the continued efficacy of many critical drugs used to treat serious infections. What if such resistant organisms could also act as altruists and "share" their resistance with sensitive cohorts without any actual genetic exchange? We competed resistant strains that differ solely in their ability to secrete a plasmid-encoded beta-lactamase. Sensitive strains were otherwise isogenic with their resistant counterparts and were either plasmid-free or contained a "Dummy" plasmid of roughly the same size as that of the resistance plasmids. Absent antibiotic selection, plasmid-free sensitive strains outperformed the plasmid-bearing strains. In the presence of ampicillin, the outcome depended on whether the resistant strain secreted its beta-lactamase (Altruist) or retained it (Selfish). In the latter case, only resistant cells survived. When beta-lactamase was secreted, some sensitive cohorts were also provided protection, with the largest fitness increase provided to plasmid-free cells. However, some Altruist strains appeared to be at a disadvantage, as a great deal of their enzyme broke off cells. Thus, additional variables must be considered when designing microbial competition experiments.

Antimicrobial Susceptibility and Plasmid Profiles of &lt;i&gt;Salmonella typhi&lt;/i&gt; isolated in Abuja, Nigeria.

Sixty clinical Salmonella spp. isolates were obtained from various health establishments in Abuja, Nigeria. These isolates were characterized using cultural, biochemical and serological assays. Antimicrobial susceptibility and plasmid analysis were used to determine the presence of plasmid-mediated multi-drug resistance. Each isolate was evaluated for susceptibility to ampicillin, chloramphenicol, gentamicin, tetracycline, cotrimoxazole, nalidixic acid, ciprofloxacin and afloxacin, using disk diffusion technique. Plasmid DNA was extracted by alkaline lysis method and fragments separated by electrophoresis on 1% agarose gel. DNA bands were visualized using ultraviolet light transilluminator and molecular weights determined. Plasmid transfer by mating was also performed using Escherichia coli K-12 as recipient. In total, 36 (60.0%) isolates were confirmed to be Salmonella typhi, of which 6 (16.7%) were fully susceptible to all microbial agents tested, while 7 (19.4%) were resistant to three or more antimicrobials including ampicillin, chloramphenicol, tetracycline, nalidixic acid and cotrimoxazole, categorized as ‘first line' drugs in the treatment of typhoid fever. However, all isolates evaluated were susceptible to ciprofloxacin and ofloxacin. Of the seven multi-drug resistant strains, 6 (85.7%) possessed plasmid sizes of 4.7 – 5.7 kilobase, suggesting that transmissible R-plasmids might be responsible for the drug resistance observed in Salmonella typhi strains in this study. Our findings show that plasmid-bearing multi drug resistant strains account for a reasonable percentage of Salmonella typhi isolates in Abuja, Nigeria. Keywords: Salmonella typhi, Plasmid, Antimicrobial resistance, typhoid fever. Journal of Medical Laboratory Sciences Vol. 14 (2) 2005: pp. 46-53

Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by Pseudomonas bacteria

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24°C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS1391 bearing plasmid pBS216 was found to be the most effective oil degrader.

Comment on: Quinolone resistance determinant qnrA3 in clinical isolates of Salmonella in 2000-2005 in Hong Kong

Sir, In their recent correspondence, Chu et al. noted the first observation of qnrA3 in salmonellae in Hong Kong, underlining the spread and diversity of plasmidic quinoloneresistance determinants worldwide. Such determinants now include qnrA1–5, qnrB and qnrS, some of which, at least, protect DNA gyrase, as well as the fluoroquinolone-acetylating variant of the aminoglycoside-modifying enzyme AAC(60)Ib, named AAC(60)Ib-cr. These proteins may influence the development of high-level resistance to at least some fluoroquinolones. In our recent article, we sought to explore the unexplained significant association between chromosomal fluoroquinolone resistance and plasmidic resistances to other antibiotic classes (such as ESBLs), which is seen in several bacterial genera. We were pleased to note that Chu et al. cited our article, but would like to clarify a misinterpretation of our hypothesis. Multiresistance plasmids can encode toxin–antitoxin systems to prevent their own loss from the cell, and some toxins target DNA gyrase. We asked whether the development of quinolone resistance by plasmid-bearing strains might be associated with such DNA gyrase-targeting toxins. We speculated on whether a cell might escape addiction to toxin–antitoxin systems by developing resistance to the toxin. Mutational events in DNA gyrase could play a role in cells escaping addiction to plasmids with gyrase-targeting toxins, and we suggested that these mutations might fortuitously confer a degree of cross-resistance to quinolones. Notably, whilst at least three qnrA variants likely originated from the chromosome of Shewanella algae, and although Qnr-type proteins inhibit/protect DNA gyrase, their precise cellular role(s) remains unknown. We would like to emphasize that we did not state, or wish to imply, that Qnr proteins could be part of a toxin–antitoxin pair as was attributed to our article by Chu et al. Rather, we postulated that gyrase ‘inhibitors/protectors’ such as Qnr might give some crossresistance to gyrase-targeting toxins in addition to the more obvious resistance to quinolones. By so doing these proteins might open a ‘window’ and influence the frequency at which cells could eject the toxin–antitoxin encoding replicons without suffering apoptosis, thereby providing the host organism with a toxin–antitoxin evasion mechanism. This might provide a competitive advantage in demanding psychrophilic aquatic environments, the natural environment of S. algae, by allowing enhanced ejection of ‘accessory’ replicons.