Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this ‘viable but nonculturable’ (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.

Recent findings on the viable but nonculturable state in pathogenic bacteria.

Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.

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The importance of the viable but non-culturable state in human bacterial pathogens.

Sources and Risk Factors for Contamination, Survival, Persistence, and Heat Resistance of Salmonella in Low-Moisture Foods

Edwardsiellosis is one of the most important bacterial diseases in fish. Scientific work on this disease started more than forty years ago and numerous workers around the world are continually adding to the knowledge of the disease. In spite of this, not a single article that reviews the enormous scientific data thus generated is available in the English language. This article briefly discusses some of the recent research on edwardsiellosis, describing the pathogen’s interaction with the host and environment, its pathogenesis and pathology as well as diagnostic, preventive and control measures.

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Edwardsiellosis in fish: a brief review

Application of flow cytometry to industrial microbial bioprocesses

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4°C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 109 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4 degrees C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10(10) to 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.

Characterization and resuscitation of viable but nonculturable Vibrio alginolyticus VIB283

In aquaculture, bacterial diseases create challenging problems because they are di⁄cult to control and often result in sudden, severe economic losses. Some of the major ¢sh pathogen bacteria cause vibriosis or haemorrhagic septicaemia with extensive skin lesions and damage internal organs such as the liver, kidneyand spleen inwildmarine ¢sh, freshwater ¢sh and other aquatic animals. Treatment of bacterial diseases is mainly based on the use of antibiotics. However, environmental concerns and the emergence of antibiotic-resistant strains of ¢sh common pathogens limit the value of these compounds and has created a need for new antimicrobial agents. The £owers of Rosa chinensis Jacq. were obtained at a local drug store (Qingdao, China) and extracted by 95% ethanol (0.2 g £owersmL 1 ethanol) at room temperature for 7 days to obtain a crude extract. The antibacterial activity of the extract against bacteria was examined by the disc diiusion method on agar plates (Nagashima, Kikuchi, Shimakura & Shiomi 2003). Eleven bacteria strains (Table1) were cultured in sterilised 2216E medium [5 g peptone, 1g yeast, 0.1g FePO4, Sinopharm Chemical Reagent, (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) 1L seawater at 33 0.8 g L 1 salinity, pH57.6] at 28 1C or LB medium [10 g peptone, 5 g yeast, 10 g NaCl, Sinopharm Chemical Reagent (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China),1L distilled water, pH57.0] at 37 1C for 24 h. The cultures were diluted with 0.85% NaCl to a concentration of 10 CFUmL 1 and 50 mL of the bacterial suspension was seeded on 2216E or LB agar plates [15 gagar L 1 distilled water or seawater, Sinopharm Chemical Reagent (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China)]. Extract (0.1mL from 20mg £owers) was applied to steriled paper discs and allowed to dry, placed on the agar plates and then incubated at 28 or 37 1C for 24 h.The bacteriawere tested in three replicates with reproducible results. Statistical analysis was performed using the commercial software program SPSS10.0. The ethanol extract of the £owers showed antibacterialactivityagainstall thetestedvibriostrains (Vibrio alginolyticusVIB 283,V. alginolyticusVIB 284,V. anguillarumCW1,V.campbelliVIB298,V.harveyiSF1,V.minicus VIB298,V. parahaemolyticus FYZ 8621.4,V. vulni¢cus VIB310)andthreenon-vibriostrainsincludingEdwardsiella tarda CW7, Aeromonas hydrophila1-2017 and A. caviae1-1960. The diameters of the growth inhibition zones ranged from 11 to 20mm when 0.1mL (20mg £owers) of theextractwasused (Table1). One kilogramof the £owers of R. chinensis Jacq. was extracted with 5 L 95% ethanol for 7 days. The crude extract was ¢ltered under reduced pressure, and then concentratedundervacuumusingarotaryevaporator. Theconcentratedethanolextract(90 g)wasthenchromatographed on a vacuum silica gel column. Elution was performed using a polarity-increasing mixture of CHCl3^MeOH. This process collected 29 fractions, whichwere tested for theantibacterialactivityagainst V. anguillarumCW1. Fractions 21^23 showed themost pronounced antibacterial activity. These fractions (10 g) were then vacuum column chromatographed Aquaculture Research, 2007, 38, 1110^1112 doi:10.1111/j.1365-2109.2007.01745.x

SHORT COMMUNICATION Antibacterial activity of gallic acid from the flowers of Rosa chinensis Jacq. against fish pathogens

Antibacterial activity of gallic acid from the flowers of Rosa chinensis Jacq. against fish pathogens

Tigecycline and carbapenems are last-resort antimicrobial agents to treat serious infections caused by multi-drug resistant bacterial pathogens. However, the co-occurrence of tigecycline and carbapenem resistance determinants challenges the clinical efficacy of these antimicrobial agents. In this study, we report the co-existence of tet(X4), blaNDM−1 and blaOXA−58 genes in the porcine Acinetobacter towneri isolate 19110F47. Sequence analysis revealed that tet(X4) gene, along with the florfenicol resistance gene floR, was flanked by three copies of IS91-like elements, which can form three different translocatable units (TUs), and were located in a 41,098-bp multidrug resistance region (MDRR) within a novel 100,354-bp genomic island (GI) region. TUs comprising floR-virD2-ISVsa3, hp-abh-tet(X4)-ISVsa3 and virD2-floR-ISVsa3-hp-abh-tet(X4)-ISVsa3 can be looped out from the chromosomal DNA and facilitate the transfer of the TU-based resistance genes into other plasmidic or chromosomal sites. In addition, the carbapenemase genes blaNDM−1 and blaOXA−58 were found on different non-conjugative multiresistance plasmids in this isolate, with the genetic contexts ISAba125-blaNDM−1-bleMBL-tnpR and ΔISAba3-blaOXA−58-ISAba3, respectively. The simultaneous occurrence of tet(X4), blaNDM−1 and blaOXA−58 in the same porcine A. towneri isolate emphasizes the importance of antimicrobial resistance surveillance in food-producing animals.

Characterization of a genomic Island carrying the tet(X4) gene in porcine Acinetobacter towneri co-harboring plasmid-borne blaNDM−1 and blaOXA−58 genes

Aijuan Li

Papers

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Characterization and resuscitation of viable but nonculturable Vibrio alginolyticus VIB283

SHORT COMMUNICATION Antibacterial activity of gallic acid from the flowers of Rosa chinensis Jacq. against fish pathogens

Antibacterial activity of gallic acid from the flowers of Rosa chinensis Jacq. against fish pathogens

Characterization of a genomic Island carrying the tet(X4) gene in porcine Acinetobacter towneri co-harboring plasmid-borne blaNDM−1 and blaOXA−58 genes