Preliminary selection study of potential probiotic bacteria from aquacultural area in Tunisia

In order to test the ability to produce antibacterial substances within marine bacteria, prior to select potential probiotics for use in shellfish farming, we targeted a large collection of bacterial isolates (132 strains), brought from the clamRuditapes decussatus and 37 reference strains. First, we proceeded to their biochemical identification and the screening of antibiotic resistance profiles. Else, we investigated their inhibitory activityin vitro against several fish and shellfish pathogens, using two methods: the double-layer agar and the direct simultaneous antagonism methods. The results showed high frequencies of inhibitory producing bacteria (IPB) within the isolates. These bacteria (25%) were aerobic mesophylic bacteria belonging to various bacterial groups: 33.7% oxidase-positive Gram-negative bacteria, 7.4%Enterobacteriaceae and 28% lactic acid bacteria. Besides this group, nine strains produced strong inhibition effect. These bacteria belonged to:Aeromonas hydrophila, Aeromonas sobria, Pseudomonas cepacia, Vibrio sp,Serratia liquefaciens andLactobacillus rhamnosus. They were active against pathogenic bacteria belonging to the genera:Aeromonas, Pseudomonas andVibrio. These potential probiotics were submitted to further investigations prior to their introduction in larval shellfish farming.     

植物病原细菌毒性调控网络的研究进展

植物病原细菌通过复杂和精细的全局性调控网络来协调多个层面的毒性决定因子。在不同的植物病原细菌中,这些全局性的毒性调控网络控制着细菌的侵染策略、存活以及在面临寄主植物防卫系统的互作环境中实现成功侵染的病程。本文详细分析了植物病原细菌4个重要属(假单胞菌属、果胶杆菌属、黄单胞菌属和雷尔氏菌属)的模式病原菌主要的毒性调控系统,包括群体感应系统、双组分调控系统、转录激活调控子以及转录后、翻译后的调控机制。在此基础上,重点评价了一些模式菌株全局性毒性调控机制的异同点,总结了一些最新的研究进展,并绘制了精细的网络调控图。这些分析表明,虽然一些相同的调控系统控制着病原菌的毒性,但是在不同种以及种下的亚种或者致病变种中这些调控机制功能各异,对于病原菌全毒性的贡献也存在着明显的差异。     

Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay,China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15–6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.     

Multidrug-resistant Opportunistic and Pathogenic Bacteria Contaminate Algerian Banknotes Currency

Currency is one of the most exchanged items in human communities as it is used daily in exchange for goods and services. It is handled by persons with different hygiene standards and can transit in different environments. Hence, money can constitute a reservoir for different types of human pathogens. This study aimed to evaluate the potential of Algerian banknotes to shelter opportunistic pathogenic and multiresistant bacteria. To that end, 200 circulating notes of four different denominations were collected from various places and analyzed for their bacterial loads and contents. Besides, predominant strains were identified and characterized by biochemical and molecular methods, and their resistance profiles against 34 antibiotics were determined. Our results indicated that 100% of the studied banknotes were contaminated with bacteria. The total bacterial concentrations were relatively high, and different bacterial groups were grown, showing important diversity. In total, 48 predominant strains were identified as belonging to 17 genera. Staphylococcus and Micrococcus were the most prevalent genera, followed by Bacillus, Pseudomonas, and Acinetobacter. Antibiotic susceptibility testing showed that all the isolates harbored resistance to at least two molecules, and worrying resistance levels were observed. These findings prove that Algerian currency harbors opportunistic multiresistant bacteria and could potentially act as a vehicle for the spread of bacterial diseases and as a reservoir for antibiotic resistance genes among the community. Therefore, no cash payment systems should be developed and generalized to minimize cash handling and subsequent potential health risks.Key words: currency, Algeria, opportunistic bacteria, antibiotic resistance, circulating resistance genes     

How do antibiotic‐producing bacteria ensure their self‐resistance before antibiotic biosynthesis incapacitates them?

Acquired antibiotic resistance among dangerous bacterial pathogens is an increasing medical problem. While in Mycobacterium tuberculosis this occurs by mutation in the genes encoding the targets for antibiotic action, other pathogens have generally gained their resistance genes by horizontal gene transfer from non‐pathogenic bacteria. The ultimate source of many of these genes is almost certainly the actinomycetes that make the antibiotics and therefore need self‐protective mechanisms to avoid suicide. How do they ensure that they are resistant at the time when intracellular antibiotic concentrations reach potentially lethal levels? In this issue of Molecular Microbiology, Tahlan et al. describe a solution to this problem in which an antibiotically inactive precursor of a Streptomyces coelicolor antibiotic induces resistance – in this example by means of a trans‐membrane export pump – so that the organism is already primed for resistance at the time when it is needed. The authors generalize their interpretation to other cases where antibiotic resistance depends on export, but it will be interesting to find out whether it could in fact apply more widely, to include the other major mechanisms of resistance: target modification and the synthesis of antibiotics via a series of chemically modified intermediates, with removal of the protective group at the time of secretion into the outside medium.     

A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria

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

A comparative genomic analysis of Xanthomonas arboricola pv. juglandis strains reveal hallmarks of mobile genetic elements in the adaptation and accelerated evolution of virulence

Xanthomonas arboricola pv. juglandis (Xaj) is the most significant aboveground walnut bacterial pathogen. Disease management uses copper-based pesticides which induce pathogen resistance. We examined the genetic repertoire associated with adaptation and virulence evolution in Xaj. Comparative genomics of 32 Xaj strains reveal the possible acquisition and propagation of virulence factors via insertion sequences (IS). Fine-scale annotation revealed a Tn3 transposon (TnXaj417) encoding copper resistance genes acquired by horizontal gene transfer and associated with adaptation and tolerance to metal-based pesticides commonly used to manage pathogens in orchard ecosystems. Phylogenomic analysis reveals IS involvement in acquisition and diversification of type III effector proteins ranging from two to eight in non-pathogenic strains, 16 to 20 in pathogenic strains, besides six other putative effectors with a reduced identity degree found mostly among pathogenic strains. Yersiniabactin, xopK, xopAI, and antibiotic resistance genes are also located near ISs or inside genomic islands and structures resembling composite transposons.     

Engineering bacteriocin‐mediated resistance against the plant pathogen Pseudomonas syringae

The plant pathogen, Pseudomonas syringae (Ps), together with related Ps species, infects and attacks a wide range of agronomically important crops, including tomato, kiwifruit, pepper, olive and soybean, causing economic losses. Currently, chemicals and introduced resistance genes are used to protect plants against these pathogens but have limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative strategies to combat bacterial disease in crops. One such strategy involves using narrow‐spectrum protein antibiotics (so‐called bacteriocins), which diverse bacteria use to compete against closely related species. Here, we demonstrate that one bacteriocin, putidacin L1 (PL1), can be expressed in an active form at high levels in Arabidopsis and in Nicotiana benthamiana in planta to provide effective resistance against diverse pathovars of Ps. Furthermore, we find that Ps strains that mutate to acquire tolerance to PL1 lose their O‐antigen, exhibit reduced motility and still cannot induce disease symptoms in PL1‐transgenic Arabidopsis. Our results provide proof‐of‐principle that the transgene‐mediated expression of a bacteriocin in planta can provide effective disease resistance to bacterial pathogens. Thus, the expression of bacteriocins in crops might offer an effective strategy for managing bacterial disease, in the same way that the genetic modification of crops to express insecticidal proteins has proven to be an extremely successful strategy for pest management. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.     

Assessment of environmental enterococci: bacterial antagonism,pathogenic capacity and antibiotic resistance

The properties of 166 environmental strains belonging to the seven enterococcal species were studied. Enterococci originated mainly from surface- and waste-waters. They were screened for the presence of enterocins, virulence factors, and antibiotic resistance. The presence of different enterocin genes (entA, entB, entP, ent31, entL50AB) was frequently observed in our enterococcal isolates, 109 strains contained at least one enterocin gene. The distribution of enterocin genes varied according to the species, the genes were present mainly in E. hirae and E. faecium. By enterocin spot assay, 10 isolates inhibited the growth of Listeria strains. To evaluate the pathogenic ability of isolates, the distribution of selected virulence genes (cylA, gelE and esp) was investigated, eleven strains were positive in some of these genes, five of them belonged to E. faecalis. Regarding the antibiotic resistance of isolates, only two strains were multiresistant and two strains (E. hirae and E. casseliflavus) were resistant to vancomycin.     

Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie

Members of the genus Aeromonas are ubiquitous in nature and have increasingly been implicated in numerous diseases of humans and other animal taxa. Although some species of aeromonads are human pathogens, their presence, density, and relative abundance are rarely considered in assessing water quality. The objectives of this study were to identify Aeromonas species within Lake Erie, determine their antibiotic resistance patterns, and assess their potential pathogenicity. Aeromonas strains were isolated from Lake Erie water by use of Aeromonas selective agar with and without tetracycline and ciprofloxacin. All isolates were analyzed for hemolytic ability and cytotoxicity against human epithelial cells and were identified to the species level by using 16S rRNA gene restriction fragment length polymorphisms and phylogenetic analysis based on gyrB gene sequences. A molecular virulence profile was identified for each isolate, using multiplex PCR analysis of six virulence genes. We demonstrated that Aeromonas comprised 16% of all culturable bacteria from Lake Erie. Among 119 Aeromonas isolates, six species were identified, though only two species (Aeromonas hydrophila and A. veronii) predominated among tetracycline- and ciprofloxacin-resistant isolates. Additionally, both of these species demonstrated pathogenic phenotypes in vitro. Virulence gene profiles demonstrated a high prevalence of aerolysin and serine protease genes among A. hydrophila and A. veronii isolates, a genetic profile which corresponded with pathogenic phenotypes. Together, our findings demonstrate increased antibiotic resistance among potentially pathogenic strains of aeromonads, illustrating an emerging potential health concern.     

Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin

Virulence properties of pathogenic bacteria, as well as resistance to antibiotics, are thought to arise through a specialization process favoured by the strong selection pressure imposed in clinical treatments. Nevertheless, in the case of opportunistic pathogens, it is unclear whether strains can be classified into virulent and non-virulent isolates. Clones of the opportunistic pathogen Pseudomonas aeruginosa do not seem to be associated to a particular biovar or pathovar, which suggests that virulence characteristics in opportunistic pathogens may already be present in environmental (non-clinical) isolates. We have explored this possibility, studying environmental isolates (mainly from oil-contaminated soils) and clinical isolates (from bacteraemia and cystic fibrosis patients) of P. aeruginosa . All environmental strains were found to actively efflux quinolones, which are synthetic antibiotics not expected to be present in the environment. These strains contained multidrug resistance determinants, were capable of invading epithelial cells and presented genes from the quorum-sensing and type III secretion systems. Some of them expressed either haemolytic or proteolytic activities or both, characteristics considered to be typical of virulent strains. All the strains tested, of clinical or environmental origin, could use alkanes (oil hydrocarbons) as a carbon source. Our results suggest that clinical and non-clinical P. aeruginosa strains might be functionally equivalent in several traits relevant for their virulence or environmental properties. Selection of clinically relevant traits, such as antibiotic resistance or cellular invasiveness, in opportunistic pathogens present in soil ecosystems is discussed.     

Ibis T5000: a universal biosensor approach for microbiology

We describe a new technology, the Ibis T5000, for the identification of pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic acid amplification to high-performance electrospray ionization mass spectrometry and base-composition analysis. The system enables the identification and quantification of a broad set of pathogens, including all known bacteria, all major groups of pathogenic fungi and the major families of viruses that cause disease in humans and animals, along with the detection of virulence factors and antibiotic resistance markers.     

A functional gene array for detection of bacterial virulence elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.     

Presence of Quorum-Sensing-Mediated Gene Regulation in Pathogenic Ice-Nucleation-Active (INA) Bacteria

Nine out of a total of 20 pathogenic ice-nucleation-active bacteria, with different levels of inducible INA, were tested and found positive for their ability to synthesize quorum-sensing (QS) signals. The bacteria were isolated from willow plants and belonged to the genera Bacillus, Erwinia, Pseudomonas and Sphingomonas. As reporter bacteria, to detect the homoserine lactone (HSL) autoinducer, Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeroginosa, Aeromonas hydrophila and Vibrio fischeri strains were used. We thus provide evidence that pathogenic ice-nucleation bacteria with inducible INA produce QS signals that in other bacteria have been shown to be in the control of genes of importance for pathogenicity.     

Genome analysis of probiotic bacteria for antibiotic resistance genes

To date, probiotic bacteria are used in the diet and have various clinical applications. There are reports of antibiotic resistance genes in these bacteria that can transfer to other commensal and pathogenic bacteria. The aim of this study was to use whole-genome sequence analysis to identify antibiotic resistance genes in a group of bacterial with probiotic properties. Also, this study followed existing issues about the importance and presence of antibiotic resistance genes in these bacteria and the dangers that may affect human health in the future. In the current study, a collection of 126 complete probiotic bacterial genomes was analyzed for antibiotic resistance genes. The results of the current study showed that there are various resistance genes in these bacteria that some of them are transferable to other bacteria. The tet(W) tetracycline resistance gene was more than other antibiotic resistance genes in these bacteria and this gene was found in Bifidobacterium and Lactobacillus. In our study, the most numbers of antibiotic resistance genes were transferred with mobile genetic elements. We propose that probiotic companies before the use of a micro-organism as a probiotic, perform an antibiotic susceptibility testing for a large number of antibiotics. Also, they perform analysis of complete genome sequence for prediction of antibiotic resistance genes.

     

Diversity of locust gut bacteria protects against pathogen invasion

Diversity–invasibility relationships were explored in the novel context of the colonization resistance provided by gut bacteria of the desert locust Schistocerca gregaria against pathogenic bacteria. Germ‐free insects were associated with various combinations of one to three species of locust gut bacteria and then fed an inoculum of the pathogenic bacterium Serratia marcescens. There was a significant negative relationship between the resulting density of Serratia marcescens and the number of symbiotic gut bacterial species present. Likewise there was a significant inverse relationship between community diversity and the proportion of locusts that harboured Serratia. Host mortality was not negatively correlated with resistance to gut‐invasion by Serratia marcescens, although there were significantly more deaths among pathogen fed germ‐free insects than tri‐associated gnotobiotes. The outcome is consistent with the predictions of community ecology theory that species‐rich communities are more resistant to invasion than species‐poor communities.     

Clinical and Environmental <Emphasis Type="Italic">Burkholderia</Emphasis> Strains: Biofilm Production and Intracellular Survival

Bacteria belonging to the Burkholderia species are important pulmonary pathogens in cystic fibrosis (CF) patients. Their ability to establish chronic and sometimes fatal infections seems linked to the quorum sensing-regulated expression of virulence factors. We examined 23 Burkholderia isolates, 19 obtained from CF patients and 4 from the environment, to evaluate their ability to form biofilm and to penetrate and replicate inside J774 macrophagic cells. Our results indicate that biofilm formation and intracellular survival are behavioral traits frequently expressed by Burkholderia strains isolated from CF patients. Successive isolates obtained from each of four chronically infected patients yielded bacteria consistently belonging to the same strain but showing increasing ability to replicate intracellularly and to produce biofilm, possibly due to in vivo bacterial microevolution driven by the selective lung environmental conditions. Protection against antimicrobials granted to burkholderiae by the expression of these two virulence factors might account for the frequent failures of antibiotic treatment in CF patients.     

Bacteriophage-mediated transduction of antibiotic resistance in enterococci

Aims: Temperate bacteriophages are bacterial viruses that transfer genetic information between bacteria. This phenomenon is known as transduction, and it is important in acquisition of bacterial virulence genes and antimicrobial resistance determinants. The aim of this study was to demonstrate the role of bacteriophages in gene transfer (antibiotic resistance) in enterococci. Methods and Results: Three bacteriophages from environmental samples isolated on pig host strains of Enterococcus gallinarum and Enterococcus faecalis were evaluated in transduction experiments. Antibiotic resistance was transferred from Ent. gallinarum to Ent. faecalis (tetracycline resistance) and from Ent. faecalis to Enterococcus faecium, Enterococcus hirae/durans and Enterococcus casseliflavus (gentamicin resistance). Conclusions: Bacteriophages play a role in transfer of antibiotic resistance determinants in enterococci. Significance and Impact of the Study: This study confirms previous suggestions on transduction in enterococci, in particular on interspecies transduction. Interspecies transduction is significant because it widens the range of recipients involved in antimicrobial resistance transfer.     

Prevalence and antibiotic resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in tropical seafood

Summary The occurrence and antibiotic resistance of Escherichia coli in tropical seafood was studied. A 3-tube MPN method was used for determining the level of faecal contamination of fresh and processed seafood. Of the 188 samples tested which included finfish, shellfish, water and ice, 155 were positive for the presence of faecal coliforms following incubation at 44.5 °C. However, E. coli was isolated from only 47% of the samples positive for faecal coliforms. The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different antibiotics including ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamycin, nalidixic acid, streptomycin and vancomycin. Seven strains were resistant to more than five antibiotics of which one was resistant to eight antibiotics. The multiple drug resistant strains harboured plasmids of varying sizes. Antibiotic susceptibility studies revealed that seafood from India contains multiple antibiotic resistant strains of E. coli which may serve as a reservoir for antibiotic resistance genes in the aquatic environment. All the strains used in this study did not harbour any virulence genes commonly associated with pathogenic E. coli, when tested by polymerase chain reaction (PCR).     

Isolation of New Delhi metallo‐β‐lactamase 1‐producing Vibrio cholerae non‐O1, non‐O139 strain carrying ctxA,st and hly genes in southern Vietnam

Vibrio cholerae non‐O1, non‐O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non‐pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG‐specific heat‐stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010–2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo‐β‐lactamase (NDM‐1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM‐1 and the bla_NDM‐1 gene was detected in those strains by PCR. Of note, one of the three NDM‐1‐producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM‐1‐producing VC_NAG strains in Vietnam.