Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Pseudomonas aeruginosa in a hydropathic facility: diversity, susceptibility and imipenem resistance mutation

Aims: To detect Pseudomonas aeruginosa in water and treatment equipment biofilms of a thermae hydropathic facility and to study antibiotic susceptibility and genetic diversity. Methods and Results: One hundred and fifty‐four planktonic isolates were obtained from 2220 water samples during 4 years. Seventy‐two biofilm isolates were obtained from 23 samples of inner parts of three inhalation equipments. Antibiotic susceptibility was determined by disc diffusion. All isolates were susceptible to tested antimicrobials, except two biofilm isolates and one planktonic isolate. Twenty‐one resistant mutants were observed (nine from biofilms), mostly with imipenem (IP) resistance (81%), by diminished expression of OprD porin, as it was observed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Random amplification polymorphic DNA showed a genetically heterogeneous population that is spread through the entire system and persistent in time. IP resistance mutation ability was spread through the population. Conclusions: The permanent assessment of Ps. aeruginosa is necessary not only in water, as expressed in official programmes, but also in equipments where biofilms are evident. Ps. aeruginosa was more prevalent in biofilm populations and presented higher ability to adapt to antibiotic pressure. Significance and Impact of the Study: Twenty‐one million people use thermae in Europe. Official microbiological quality control programmes only consider water surveillance. Present study proves the need of a review on current official programmes.     

Genome Analysis of Environmental and Clinical P. aeruginosa Isolates from Sequence Type-1146

The genomes of Pseudomonas aeruginosa isolates of the new sequence type ST-1146, three environmental (P37, P47 and P49) and one clinical (SD9) isolates, with differences in their antibiotic susceptibility profiles have been sequenced and analysed. The genomes were mapped against P. aeruginosa PAO1-UW and UCBPP-PA14. The allelic profiles showed that the highest number of differences were in “Related to phage, transposon or plasmid” and “Secreted factors” categories. The clinical isolate showed a number of exclusive alleles greater than that for the environmental isolates. The phage Pf1 region in isolate SD9 accumulated the highest number of nucleotide substitutions. The ORF analysis of the four genomes assembled de novo indicated that the number of isolate-specific genes was higher in isolate SD9 (132 genes) than in isolates P37 (24 genes), P47 (16 genes) and P49 (21 genes). CRISPR elements were found in all isolates and SD9 showed differences in the spacer region. Genes related to bacteriophages F116 and H66 were found only in isolate SD9. Genome comparisons indicated that the isolates of ST-1146 are close related, and most genes implicated in pathogenicity are highly conserved, suggesting a genetic potential for infectivity in the environmental isolates similar to the clinical one. Phage-related genes are responsible of the main differences among the genomes of ST-1146 isolates. The role of bacteriophages has to be considered in the adaptation processes of isolates to the host and in microevolution studies.     

Determination of the Efflux Pump-Mediated Resistance Prevalence in Pseudomonas aeruginosa, Using an Efflux Pump Inhibitor

In Gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl β-naphtylamide (PAβN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256–0.25 μg/ml) with a fixed concentration of PAβN (50 μg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAβN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAβN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.     

Two Pseudomonas aeruginosa clonal groups belonging to the PA14 clade are indigenous to the Churince system in Cuatro Ciénegas Coahuila,México

Pseudomonas aeruginosa is a widely distributed environmental bacterium but is also an opportunistic pathogen that represents an important health hazard due to its high intrinsic antibiotic resistance and its production of virulence factors. The genetic structure of P. aeruginosa populations using whole genome sequences shows the existence of three clades, one of which (PA7 clade) has a higher genetic diversity. These three clades include clinical and environmental isolates that are very diverse in terms of geographical origins and isolation date. Here, we report the characterization of two distinct clonal P. aeruginosa groups that form a part of the PA14 clade (clade 2) sampled from the Churince system in Cuatro Ciénegas Basin (CCB). One of the clonal groups that we report here was isolated in 2011 (group 2A) and was displaced by the other clonal group (2B) in 2015. Both Churince groups are unable to produce pyoverdine but can produce other virulence-associated traits. The existence of these unique P. aeruginosa clonal groups in the Churince system is of ecological and evolutionary significance since the microbiota of this site is generally very distinct from other lineages, and this is the first time that a population of P. aeruginosa has been found in CCB.     

Crosstalk between Pseudomonas aeruginosa antibiotic resistance and virulence mediated by phenylethylamine

Multidrug efflux pumps are among the main Pseudomonas aeruginosa antibiotic-resistance determinants. Besides, efflux pumps are also involved in other relevant activities of bacterial physiology, including the quorum sensing-mediated regulation of bacterial virulence. Nevertheless, despite the relevance of efflux pumps in bacterial physiology, their interconnection with bacterial metabolism remains obscure. The effect of several metabolites on the expression of P. aeruginosa efflux pumps, and on the virulence and antibiotic resistance of this bacterium, was studied. Phenylethylamine was found to be both inducer and substrate of MexCD-OprJ, an efflux pump involved in P. aeruginosa antibiotic resistance and in extrusion of precursors of quorum-sensing signals. Phenylethylamine did not increase antibiotic resistance; however, the production of the toxin pyocyanin, the tissue-damaging protease LasB and swarming motility were reduced in the presence of this metabolite. This decrease in virulence potential was mediated by a reduction of lasI and pqsABCDE expression, which encode the proteins that synthesise the signalling molecules of two quorum-sensing regulatory pathways. This work sheds light on the interconnection between virulence and antibiotic-resistance determinants, mediated by bacterial metabolism, and points to phenylethylamine as an anti-virulence metabolite to be considered in the study of therapies against P. aeruginosa infections.     

Multiple drug resistance and strength of attachment to surfaces in Pseudomonas aeruginosa isolates

Aims: To investigate the presence of a relationship between the strength of attachment of Pseudomonas aeruginosa to stainless steel surfaces and their observed multiple drug resistance. Methods and Results: Multiple drug resistance of clinical and environmental isolates of Ps. aeruginosa was evaluated using disc diffusion method. The blot succession technique was used to quantify the strength of attachment of Ps. aeruginosa isolates. Different multiple drug–resistant Ps. aeruginosa isolates exhibited variable attachment strength. Although the highest multiple drug–resistant clinical isolate was shown to have the least attachment strength among clinical isolates, a weak correlation was found between attachment strength and multiple resistance among our investigated Ps. aeruginosa isolates. Conclusions: There is a weak correlation between multiple drug resistance and strength of attachment to stainless steel surfaces. Significance and Impact of the Study: Even low‐resistant Ps. aeruginosa could have the potential of attaching firmly to surfaces and forming biofilm.     

Characterisation of potential virulence markers in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from drinking water

Pseudomonas aeruginosa isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, hemaglutinins, cytotoxins and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics, human serum sensitivity and the survival of P. aeruginosa isolates in a chlorinated environment were also examined. Of the 30 isolates tested, 16 possessed the capacity to adhere to abiotic surfaces, and 28 to adhere to epithelial cells; 30 were capable of producing hemolysins, 27 produced cytotoxins, 9 hemagglutinins, and 18 were classified as serum-resistant. For the lowest concentration of chlorine (0.2 mg/l) tested, no killing of biofilm bacteria could be discerned, even after prolonged exposure to the agent. Although all the drinking water isolates were susceptible to aztreonam, cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, piperacillin-tazobactam, and polymyxin, the P. aeruginosa isolates were resistant to one or more antibiotics. The increasing prevalence of resistance in the isolates from environmental sources may have important therapeutic implications. A notable proportion of the P. aeruginosa isolates from drinking water were able to develop virulence factors, and the incidence of virulence properties was not statistically different among the three sources. A more extensive study of the virulence properties of this bacterium by toxic assays on animals should be explored. Still more interesting would be toxicity assays on immuno-deficient animals with isolates from drinking water in order to better understand the health risk these bacteria may present.     

Spatio-temporal analysis of infra-specific genetic variations among a Pseudomonas aeruginosa water network hospital population: invasion and selection of clonal complexes

Aims: To investigate infra-specific spatio-temporal dynamics of a hospital water network Pseudomonas aeruginosa population. To infer the origin of water network isolates and assess their potential health hazard. Methods and Results: 168 P. aeruginosa strains were isolated from tap waters and swabs of tap nozzle aerators of a hospital unit, over 2 years, and from rectal swabs and nosocomial infections. Genetic diversity among this collection was assessed by pulsed field gel electrophoresis of SpeI restricted genomic DNA. Virulence gene sets, biofilm properties, and hypochlorite resistance were analysed. Exactly 68% of the water samples and 74% of the tap nozzle aerators harboured P. aeruginosa. The strains were divided into 22 clonal lineages, with one dominant clone shown to have been involved in a nosocomial infection. Conclusions: An important turnover among the P. aeruginosa hospital population was observed. Some clonal lineages were found to persist, spread in the unit, and diversify into clonal complexes. Rectal carriage appeared an important source of contamination of the water network. Significance and Impact of the Study: High P. aeruginosa infra-specific population diversity suggested a broad ability in colonizing water networks but persistence analysis indicated a strong selection leading to the emergence of dominant clones.     

Trade‐off between antibiotic resistance and biological fitness in Acinetobacter sp. strain DR1

Rifampicin, a bactericidal antibiotic drug, is routinely used to make an environmental recipient selective in laboratory‐conjugation experiments. We noticed, inadvertently, that the rifampicin‐resistant Acinetobacter sp. strain DR1, a recently discovered hexadecane‐degrading environmental isolate, exhibited a substantial loss of quorum sensing signalling. The domesticated ampicillin‐resistant strain, DR1, evidenced more dramatic phenotypic changes than were observed in the rifampicin‐resistant cells: a complete loss of quorum sensing, a loss in swimming and swarming motilities, poor fimbrial expression, increased rigidity in membrane fatty acid composition and reduced hexadecane degradation capability. Interestingly, the motility of strain DR1 grown adjacent to a streptomycin‐producing Streptomyces griceus was permanently abrogated, where this change was heritable and other phenotypic changes could not be detected. In this study, we have reported for the first time that the in situ acquisition of antibiotic resistance may reduce biological fitness, including losses in the production of quorum sensing signals, motility and substrate utilization, and each antibiotic is associated with different degrees of phenotypic and genetic alterations. Our data also suggested that the domestication of environmental isolates should be approached with caution, as there are phenotypic variations in antibiotic‐resistant cells that might not be noticeable unless all possible phenotypic assays are conducted.     

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Isolation and identification of methylobacterium species from the tap water in hospitals in Japan and their antibiotic susceptibility

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90 = 1 microg/ml) and tetracycline (MIC90 = 8 microg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.     

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections

Aims: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, bla_OXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla_OXA‐31 in a canine Ps. aeruginosa isolate.     

The occurrence of Pseudomonas spp. in surface water and in tap water as determined on citrate media

Citrate-utilizing bacteria were counted in 289 samples of tap water derived from either surface water or ground water and in 32 samples of raw or partially treated surface water by using media containing ferric ammonium citrate as the carbon and energy source.The citrate-utilizing bacteria constituted only small minorities of the colony counts on Lab-Lemco agar at 25 C in both tap water and surface water.A total of 1071 isolates were obtained, of which 979 were able to utilize citrate. Characterization of the citrate-utilizing isolates revealed that 90% of these bacteria were arginine dihydrolase-positive and belonged to the genera Pseudomonas (84.3%) and Aeromonas (5.7%).The genus Pseudomonas was represented by fluorescent pseudomonads (66.3%), non-fluorescent glucose-utilizing pseudomonads (12.1%) and P. alcaligenes (5.9%). None of the isolates was identified as P. aeruginosa.It is suggested that the pseudomonads and the aeromonads are not adapted to low substrate concentrations. Enumeration of the citrate-utilizing bacteria in tap water therefore may give information on the efficiency of water treatment techniques as regards the substrate removal.     

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application

Aims: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid‐associated genetic markers in soil following manure application to an agricultural field. Methods and Results: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10β for subsequent characterization. We isolated and DNA sequenced a 22 102‐bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. Conclusions: The pMC2 plasmid has a tetracycline‐resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. Significance and Impact of the Study: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.