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A liquid medium permitting anaerobic growth of Neisseria gonorrhoeae
Journal of Microbiological Methods 79 (2009) 364–366
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Note
A liquid medium permitting anaerobic growth of Neisseria gonorrhoeae Jeremy James Wade ⁎, Michelle Angela Graver Health Protection Agency London Region Laboratory, Medical Microbiology, King's College Hospital, Denmark Hill, London, SE5 9RS, United Kingdom
a r t i c l e
i n f o
Article history: Received 19 July 2009 Received in revised form 11 September 2009 Accepted 18 September 2009 Available online 29 September 2009
a b s t r a c t Neisseria gonorrhoeae will grow in an anaerobic atmosphere if provided with nitrite as a terminal electron acceptor, and it is increasingly apparent that this important pathogen may grow anaerobically in vivo. By modifying a previously described chemically-defined medium we have produced a liquid medium capable of supporting growth of N. gonorrhoeae under strictly anaerobic conditions. © 2009 Elsevier B.V. All rights reserved.
Keywords: Anaerobic Gonorrhoea Growth Liquid Medium Neisseria
Neisseria gonorrhoeae causes gonorrhoea, a common sexuallytransmitted infection with potentially serious sequelae including pelvic inflammatory disease and infertility. N. gonorrhoeae is fastidious: growth requires enriched media, such as chocolate agar, and is usually attained for diagnostic and research purposes in an atmosphere enriched with CO2. Although previously considered incapable of anaerobic growth (James-Holmquest et al., 1973), it was shown that N. gonorrhoeae can grow in an anaerobic atmosphere if nitrite is provided as a terminal electron acceptor (Knapp and Clark, 1984). Further, it is increasingly apparent that N. gonorrhoeae grows anaerobically in vivo: gonococcus mixed with obligate anaerobes may be recovered from clinical material and N. gonorrhoeae proteins induced anaerobically include a nitrite reductase (Clark et al., 1987), antibodies to which were demonstrated in sera of each of six patients with uncomplicated gonococcal infection, pelvic inflammatory disease or disseminated gonococcal infection (Clark et al., 1988). The regulation of genes conferring ability to grow anaerobically has been studied extensively (Householder et al., 1999; Lissenden et al., 2000; Isabella et al., 2008). The realisation that N. gonorrhoeae may grow anaerobically during infection has implications for the in vitro study of this pathogen. Here we describe an adaptation of a previously published defined liquid medium (GW medium; Wade and Graver, 2007), that permits anaerobic growth of N. gonorrhoeae. Our intention in designing GW medium was to provide a chemically-defined and clear liquid medium for the study of the gonococcus which, compared
⁎ Corresponding author. Tel.: +44 203 2993033; fax: +44 203 2993404. E-mail address: [email protected] (J.J. Wade). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.09.018
to other published defined media, is easy and inexpensive to prepare. The anaerobic version described herein is equally simple to prepare. We used a panel of N. gonorrhoeae strains with various nutritional requirements as previously described (Wade and Graver, 2007). These are clinical isolates from patients attending a genitourinary medicine at King's College Hospital, recovered on standard selective media and stored at −70 °C. Auxotyping was by the method of Copley and Egglestone (1983) and auxotypes were denoted as non-requiring (NR) or as a requirement for one or more of arginine (A), hypoxanthine (H), proline (P), ornithine (O) or uracil (U). The panel of 21 strains included two representatives – distinguishable by serotyping using the Phadebact GC Serovar test (Boule Diagnostics AB, Stockholm, Sweden) – of each of the following auxotypes: PA, NR, P, AOU, PAOU, AH, A, AHU, H and PAH, and N. gonorrhoeae NCTC 8375. Fusobacterium nucleatum ATCC 25586 was included as an indicator of anaerobiosis. Achieving anaerobic growth of N. gonorrhoeae required the following changes to GW medium: exclusion of sodium acetate and lactate, reduction of glucose concentration, and the addition of ‘Oxyrase® for Broth’ (Oxyrase Inc, Ohio, USA). This anaerobic version of the medium – hereafter denoted GW-An medium – therefore required preparation of 1 L of normal strength M199 cell culture medium (commercially available with Earle's salts but without L-glutamine, phenol red or sodium bicarbonate; e.g. product number M3769 from Sigma-Aldrich, Poole, UK) in distilled water. To this, a further 500 mL distilled water was added plus (g; final molarity): glucose (2.0; 7.4 mM), ammonium bicarbonate (2.0; 17 mM), L-glutamine (0.75; 3.4 mM), spermidine (0.2; 919 μM), Larginine (0.1; 383 μM), hypoxanthine (0.05; 245 μM), uracil (0.05; 298 μM), oxaloacetate (0.05; 252 μM), thiamine hydrochloride (0.05; 99 μM), L-ornithine (0.01; 39 μM), and nicotinamide adenine dinucleotide (0.01; 10 μM). Hypoxanthine and uracil were dissolved in 2–3 mL 1 M NaOH. All reagents were from Sigma-Aldrich, Poole, UK. The pH was
J.J. Wade, M.A. Graver / Journal of Microbiological Methods 79 (2009) 364–366
adjusted to 6.5 with 1 M HCl and the medium sterilized through a 0.22 μm filter (‘Nalgene’, Thermo Fisher Scientific, Roskilde, Denmark). Oxyrase for Broth was added at a final concentration of 20 mL/L (see below). Inocula were prepared from overnight growth on chocolate agar in 5% CO2 at 37 °C for N. gonorrhoeae, or from fastidious anaerobe agar incubated anaerobically overnight at 37 °C for F. nucleatum (media from Oxoid, Basingstoke, UK). For each bacterium, a 25 mL volume of base medium (containing all components other than Oxyrase) was inoculated with 300 μL of a 10− 3 dilution of a 0.5 McFarland density standard in phosphate-buffered saline (PBS) pH 7.4; from this, three 7 mL aliquots in 7 mL sterile plastic bijoux (Bibby Sterilin Ltd, Pontypridd, UK) were prepared and time 0 counts performed using the remnant on chocolate agar or fastidious anaerobe agar, as appropriate. Bacteria were enumerated using a spiral plater (Don Whitley, Shipley, UK) and colony counts done manually or using an aCOLyte colony counter (Don Whitley, Shipley, UK). The spiral plater dispensed 50 μL per plate giving a lower limit for colony counts – on undiluted material – of 20 CFU/mL. Uninoculated controls included two 7 mL aliquots of medium with methylene blue and two 7 mL aliquots with resazurin sodium, both at 0.002 g/L. Finally, Oxyrase 150 μL was added to all 7 mL volumes other than one of each of the pairs of reduction indicator controls. The final headspace above the liquid in each bijoux was approximately 1 mL. Bijoux lids were tightly closed and all incubated at 37 °C. At times 20, 25 and 30 chemical indicator controls with and without Oxyrase were examined to confirm anaerobiosis. At the same time intervals (using one of the 7 mL triplicates for each time point) counts were done after vortexing for 5 s, on serial 10− 2 dilutions in PBS on chocolate agar for N. gonorrhoeae or fastidious anaerobe agar for F. nucleatum using a spiral plater. Aliquots were also removed for Gram stain after vortexing. Experiments were done at least in triplicate. All plates were incubated at 37 °C for 48–72 h before colonies were counted: the chocolate agar plates for N. gonorrhoeae in 5% CO2 and the fastidious anaerobe agar plates for F. nucleatum in anaerobic jars. Indicators confirmed that anaerobiosis was maintained to at least 30 h and counts (median log10 CFU/mL) for the obligate anaerobe F. nucleatum at times 0, 20, 25 and 30 h were 3.03, 7.98, 8.36 and 8.27, respectively. Twelve of the strains produced consistent growth in each of initial triplicates. Of the 9 that failed to produce growth curves, only 4 failed to give >1 log increase in growth by 30 h in one or more experiments. Eight strains required one or more additional experiments using a 300 μL inoculum to achieve reproducible growth curves growth thrice. For a further strain, an AOU auxotype, this required increasing the volume of inoculum to 400 μL in one experiment of a triplicate. For these 9 strains the time 0 counts for replicate experiments that produced growth (and are therefore included in Fig. 1) were compared with those that subsequently yielded poor or
Fig. 1. Growth of 21 distinguishable strains of N. gonorrhoeae in 7 mL volumes of GWAn medium; median values for triplicate experiments for each strain denoted by boxand-whisker plot showing median, inter-quartile range and range.
365
anomalous growth. The median log10 CFU/mL (inter-quartile range; range) was 3.13 (2.60–3.26; 2.08–3.45) for the former, and 3.13 (2.81–3.31; 1.78–3.58) for the latter. Overall, the median log10 CFU/mL count (inter-quartile range; range) for the 21 N. gonorrhoeae at 0, 20, 25 and 30 h were 3.13 (2.98– 3.21; 2.08–3.35), 6.33 (5.92–6.60; 4.08–6.89), 6.77 (6.36–6.98; 4.30– 7.20) and 6.90 (6.76–7.03; 4.45–7.16), respectively (Fig. 1). The limitations of this study are those described previously (Wade and Graver, 2007): the panel of strains is small (from an epidemiological perspective) and may not be representative; although dilutions were vortexed throughout (and clumping not noted on microscopy) we assume that one colony is the product of one bacterium; bacteria in our inocula were not washed and, despite substantial dilution, there is the possibility of carry-over of nutrients from chocolate agar. Our intention was to demonstrate that the medium described would support the growth of N. gonorrhoeae anaerobically, rather than to present growth curves and calculated growth rates. For this reason we chose a limited number of predetermined time points. We sought to determine the smallest inocula that would permit growth although this is unlikely to reflect how the medium may be used by others. It is impossible to predict the exact inoculum at the outset of each experiment as, even with the use of a turbidity standard, there is variability in the inocula. Indeed, the range of time 0 counts reveals a greater than 1 log variation (2.08–3.35). Experience has shown us that some strains may give lower counts than others despite identical inocula prepared using the same turbidity standard. For the strains that required retesting, the similarity between the time 0 counts for experiments that did yield growth and for those that did not, suggests that these inocula are likely to approximate to the lowest that can successfully initiate such growth, and therefore higher than those permitting growth in the original GW medium (median 80 CFU/mL; Wade and Graver, 2007). Although the rate of growth of N. gonorrhoeae in GW-An medium appears lower than in GW medium, there are differences not only in composition of the media but also in growth conditions: in our earlier report growth was in 50 mL GW medium in 5% CO2 agitated at 100 rpm on an orbital shaker. We believe there are intrinsic strain-dependent differences in growth rate despite provision of all the substrates necessary to satisfy the auxotrophic requirements of all strains tested. Comparing growth rates between time zero and time 20 h in the two studies (results not shown) we find that of the five slowest growing auxotypes in GW medium in CO2, four (including both AHU strains) are amongst the five slowest growing in the present study; similarly, two strains (auxotypes A and PAOU) were amongst the five most rapid growers in both studies. The ability to study N. gonorrhoeae growing anaerobically will advance our understanding of the pathogenicity of a bacterium of major public health significance. To date, anaerobic growth of N. gonorrhoeae has been achieved as a halo around sodium nitriteimpregnated discs (Knapp and Clark, 1984; Frangipane and Rest, 1992), on nitrite-containing media (Lissenden et al., 2000) or by the addition of pulses of nitrite to undefined liquid medium (Overton et al., 2006). ‘Oxyrase for Broth’ is a commercially-available biocatalytic oxygen-reducing agent containing Escherichia coli membrane fragments bearing enzymes capable of removing dissolved oxygen from liquid media within an hour of incubation at 37 °C and preventing subsequent oxygen ingress, thereby permitting the growth of strict anaerobes (Adler and Spadey, 1997). The 4.9 log increase in F. nucleatum counts over the initial 20 h period, and growth thereafter, confirm anaerobiosis; oxygen ingress from the minimal headspace was removed. The Oxyrase E. coli membrane fragments retain the ability to reduce nitrate (results not shown) and may provide adequate nitrite from the ferric nitrate present in commercially-available M199 medium to permit the growth demonstrated. In preliminary experiments additional nitrate or nitrite did
366
J.J. Wade, M.A. Graver / Journal of Microbiological Methods 79 (2009) 364–366
not increase growth rates (results not shown). The use of Oxyrase means that GW-An medium, unlike GW medium, is not strictly chemically defined. Further, the presence of E. coli membrane fragments and their associated enzymes may complicate the study of subcellular components of N. gonorrhoeae, though repeated centrifugation and washing of bacteria may reduce or remove this problem. It should be noted that, although lactate was omitted from GW-An medium, Oxyrase contains lactate as an enzyme substrate and this can be used by N. gonorrhoeae as an energy source. We are currently determining whether anaerobic growth of N. gonorrhoeae can be achieved when Oxyrase is separated from the basal medium by a dialysis membrane. The anaerobic growth of N. gonorrhoeae in the liquid medium described here – which is based on a cell culture medium – will simulate more closely the growth of this bacterium in anatomical relevant sites with a low partial pressure of oxygen, such as the female genital tract, than currently available solid or liquid bacteriological media. The medium described here offers a novel way to study growth of N. gonorrhoeae and its interactions with other bacteria under anaerobic conditions.
References Adler, H., Spadey, G., 1997. The use of microbial membranes to achieve anaerobiosis. J. Rapid Methods Automat. Microb. 5, 1–12.
Clark, V.L., Campbell, L.A., Palermo, D.A., Evans, T.M., Klimpel, K.W., 1987. Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae. Infect. Immun. 55, 1359–1364. Clark, V.L., Knapp, J.S., Thompson, S., Klimpel, K.W., 1988. Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb. Pathog. 5, 381–390. Copley, C.G., Egglestone, S.I., 1983. Auxotyping of Neisseria gonorrhoeae isolated in the United Kingdom. J. Med. Microbiol. 16, 295–302. Frangipane, J.V., Rest, R.F., 1992. Anaerobic growth of gonococci does not alter their opa-mediated interactions with human neutrophils. Infect. Immun. 60, 1793–1799. Householder, T.C., Belli, W.A., Lissenden, S., Cole, J.A., Clark, V.L., 1999. cis- and transacting elements involved in regulation of aniA, the gene encoding the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae. J. Bact. 181, 541–551. Isabella, V., Wright, L.F., Barth, K., Spence, J.M., Grogan, S., Genco, C.A., Clark, V.L., 2008. cisand trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae. Microbiology 154, 226–239. James-Holmquest, A.N., Wende, R.D., Mudd, R.L., Williams, R.P., 1973. Comparison of atmospheric conditions for culture of clinical specimens of Neisseria gonorrhoeae. Appl. Microbiol. 26, 466–469. Knapp, J.S., Clark, V.L., 1984. Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction. Infect. Immun. 46, 176–181. Lissenden, S., Mohan, S., Overton, T., Regan, T., Crooke, H., Cardinale, J.A., Householder, T.C., Adams, P., O'Conner, C.D., Clark, V.L., Smith, H., Cole, J.A., 2000. Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. Mol. Microbiol. 37, 839–855. Overton, T.W., Whitehead, R., Li, Y., Snyder, L.A.S., Saunders, N.J., Smith, H., Cole, J.A., 2006. Coordinated regulation of the Neisseria gonorrhoeae-truncated denitrification pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ– NarP. J. Biol. Chem. 44, 33115–33126. Wade, J.J., Graver, M.A., 2007. A fully defined, clear and protein-free liquid medium permitting dense growth of Neisseria gonorrhoeae from very low inocula. FEMS Microb. Lett. 273, 35–37.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Note
A liquid medium permitting anaerobic growth of Neisseria gonorrhoeae Jeremy James Wade ⁎, Michelle Angela Graver Health Protection Agency London Region Laboratory, Medical Microbiology, King's College Hospital, Denmark Hill, London, SE5 9RS, United Kingdom
a r t i c l e
i n f o
Article history: Received 19 July 2009 Received in revised form 11 September 2009 Accepted 18 September 2009 Available online 29 September 2009
a b s t r a c t Neisseria gonorrhoeae will grow in an anaerobic atmosphere if provided with nitrite as a terminal electron acceptor, and it is increasingly apparent that this important pathogen may grow anaerobically in vivo. By modifying a previously described chemically-defined medium we have produced a liquid medium capable of supporting growth of N. gonorrhoeae under strictly anaerobic conditions. © 2009 Elsevier B.V. All rights reserved.
Keywords: Anaerobic Gonorrhoea Growth Liquid Medium Neisseria
Neisseria gonorrhoeae causes gonorrhoea, a common sexuallytransmitted infection with potentially serious sequelae including pelvic inflammatory disease and infertility. N. gonorrhoeae is fastidious: growth requires enriched media, such as chocolate agar, and is usually attained for diagnostic and research purposes in an atmosphere enriched with CO2. Although previously considered incapable of anaerobic growth (James-Holmquest et al., 1973), it was shown that N. gonorrhoeae can grow in an anaerobic atmosphere if nitrite is provided as a terminal electron acceptor (Knapp and Clark, 1984). Further, it is increasingly apparent that N. gonorrhoeae grows anaerobically in vivo: gonococcus mixed with obligate anaerobes may be recovered from clinical material and N. gonorrhoeae proteins induced anaerobically include a nitrite reductase (Clark et al., 1987), antibodies to which were demonstrated in sera of each of six patients with uncomplicated gonococcal infection, pelvic inflammatory disease or disseminated gonococcal infection (Clark et al., 1988). The regulation of genes conferring ability to grow anaerobically has been studied extensively (Householder et al., 1999; Lissenden et al., 2000; Isabella et al., 2008). The realisation that N. gonorrhoeae may grow anaerobically during infection has implications for the in vitro study of this pathogen. Here we describe an adaptation of a previously published defined liquid medium (GW medium; Wade and Graver, 2007), that permits anaerobic growth of N. gonorrhoeae. Our intention in designing GW medium was to provide a chemically-defined and clear liquid medium for the study of the gonococcus which, compared
⁎ Corresponding author. Tel.: +44 203 2993033; fax: +44 203 2993404. E-mail address: [email protected] (J.J. Wade). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.09.018
to other published defined media, is easy and inexpensive to prepare. The anaerobic version described herein is equally simple to prepare. We used a panel of N. gonorrhoeae strains with various nutritional requirements as previously described (Wade and Graver, 2007). These are clinical isolates from patients attending a genitourinary medicine at King's College Hospital, recovered on standard selective media and stored at −70 °C. Auxotyping was by the method of Copley and Egglestone (1983) and auxotypes were denoted as non-requiring (NR) or as a requirement for one or more of arginine (A), hypoxanthine (H), proline (P), ornithine (O) or uracil (U). The panel of 21 strains included two representatives – distinguishable by serotyping using the Phadebact GC Serovar test (Boule Diagnostics AB, Stockholm, Sweden) – of each of the following auxotypes: PA, NR, P, AOU, PAOU, AH, A, AHU, H and PAH, and N. gonorrhoeae NCTC 8375. Fusobacterium nucleatum ATCC 25586 was included as an indicator of anaerobiosis. Achieving anaerobic growth of N. gonorrhoeae required the following changes to GW medium: exclusion of sodium acetate and lactate, reduction of glucose concentration, and the addition of ‘Oxyrase® for Broth’ (Oxyrase Inc, Ohio, USA). This anaerobic version of the medium – hereafter denoted GW-An medium – therefore required preparation of 1 L of normal strength M199 cell culture medium (commercially available with Earle's salts but without L-glutamine, phenol red or sodium bicarbonate; e.g. product number M3769 from Sigma-Aldrich, Poole, UK) in distilled water. To this, a further 500 mL distilled water was added plus (g; final molarity): glucose (2.0; 7.4 mM), ammonium bicarbonate (2.0; 17 mM), L-glutamine (0.75; 3.4 mM), spermidine (0.2; 919 μM), Larginine (0.1; 383 μM), hypoxanthine (0.05; 245 μM), uracil (0.05; 298 μM), oxaloacetate (0.05; 252 μM), thiamine hydrochloride (0.05; 99 μM), L-ornithine (0.01; 39 μM), and nicotinamide adenine dinucleotide (0.01; 10 μM). Hypoxanthine and uracil were dissolved in 2–3 mL 1 M NaOH. All reagents were from Sigma-Aldrich, Poole, UK. The pH was
J.J. Wade, M.A. Graver / Journal of Microbiological Methods 79 (2009) 364–366
adjusted to 6.5 with 1 M HCl and the medium sterilized through a 0.22 μm filter (‘Nalgene’, Thermo Fisher Scientific, Roskilde, Denmark). Oxyrase for Broth was added at a final concentration of 20 mL/L (see below). Inocula were prepared from overnight growth on chocolate agar in 5% CO2 at 37 °C for N. gonorrhoeae, or from fastidious anaerobe agar incubated anaerobically overnight at 37 °C for F. nucleatum (media from Oxoid, Basingstoke, UK). For each bacterium, a 25 mL volume of base medium (containing all components other than Oxyrase) was inoculated with 300 μL of a 10− 3 dilution of a 0.5 McFarland density standard in phosphate-buffered saline (PBS) pH 7.4; from this, three 7 mL aliquots in 7 mL sterile plastic bijoux (Bibby Sterilin Ltd, Pontypridd, UK) were prepared and time 0 counts performed using the remnant on chocolate agar or fastidious anaerobe agar, as appropriate. Bacteria were enumerated using a spiral plater (Don Whitley, Shipley, UK) and colony counts done manually or using an aCOLyte colony counter (Don Whitley, Shipley, UK). The spiral plater dispensed 50 μL per plate giving a lower limit for colony counts – on undiluted material – of 20 CFU/mL. Uninoculated controls included two 7 mL aliquots of medium with methylene blue and two 7 mL aliquots with resazurin sodium, both at 0.002 g/L. Finally, Oxyrase 150 μL was added to all 7 mL volumes other than one of each of the pairs of reduction indicator controls. The final headspace above the liquid in each bijoux was approximately 1 mL. Bijoux lids were tightly closed and all incubated at 37 °C. At times 20, 25 and 30 chemical indicator controls with and without Oxyrase were examined to confirm anaerobiosis. At the same time intervals (using one of the 7 mL triplicates for each time point) counts were done after vortexing for 5 s, on serial 10− 2 dilutions in PBS on chocolate agar for N. gonorrhoeae or fastidious anaerobe agar for F. nucleatum using a spiral plater. Aliquots were also removed for Gram stain after vortexing. Experiments were done at least in triplicate. All plates were incubated at 37 °C for 48–72 h before colonies were counted: the chocolate agar plates for N. gonorrhoeae in 5% CO2 and the fastidious anaerobe agar plates for F. nucleatum in anaerobic jars. Indicators confirmed that anaerobiosis was maintained to at least 30 h and counts (median log10 CFU/mL) for the obligate anaerobe F. nucleatum at times 0, 20, 25 and 30 h were 3.03, 7.98, 8.36 and 8.27, respectively. Twelve of the strains produced consistent growth in each of initial triplicates. Of the 9 that failed to produce growth curves, only 4 failed to give >1 log increase in growth by 30 h in one or more experiments. Eight strains required one or more additional experiments using a 300 μL inoculum to achieve reproducible growth curves growth thrice. For a further strain, an AOU auxotype, this required increasing the volume of inoculum to 400 μL in one experiment of a triplicate. For these 9 strains the time 0 counts for replicate experiments that produced growth (and are therefore included in Fig. 1) were compared with those that subsequently yielded poor or
Fig. 1. Growth of 21 distinguishable strains of N. gonorrhoeae in 7 mL volumes of GWAn medium; median values for triplicate experiments for each strain denoted by boxand-whisker plot showing median, inter-quartile range and range.
365
anomalous growth. The median log10 CFU/mL (inter-quartile range; range) was 3.13 (2.60–3.26; 2.08–3.45) for the former, and 3.13 (2.81–3.31; 1.78–3.58) for the latter. Overall, the median log10 CFU/mL count (inter-quartile range; range) for the 21 N. gonorrhoeae at 0, 20, 25 and 30 h were 3.13 (2.98– 3.21; 2.08–3.35), 6.33 (5.92–6.60; 4.08–6.89), 6.77 (6.36–6.98; 4.30– 7.20) and 6.90 (6.76–7.03; 4.45–7.16), respectively (Fig. 1). The limitations of this study are those described previously (Wade and Graver, 2007): the panel of strains is small (from an epidemiological perspective) and may not be representative; although dilutions were vortexed throughout (and clumping not noted on microscopy) we assume that one colony is the product of one bacterium; bacteria in our inocula were not washed and, despite substantial dilution, there is the possibility of carry-over of nutrients from chocolate agar. Our intention was to demonstrate that the medium described would support the growth of N. gonorrhoeae anaerobically, rather than to present growth curves and calculated growth rates. For this reason we chose a limited number of predetermined time points. We sought to determine the smallest inocula that would permit growth although this is unlikely to reflect how the medium may be used by others. It is impossible to predict the exact inoculum at the outset of each experiment as, even with the use of a turbidity standard, there is variability in the inocula. Indeed, the range of time 0 counts reveals a greater than 1 log variation (2.08–3.35). Experience has shown us that some strains may give lower counts than others despite identical inocula prepared using the same turbidity standard. For the strains that required retesting, the similarity between the time 0 counts for experiments that did yield growth and for those that did not, suggests that these inocula are likely to approximate to the lowest that can successfully initiate such growth, and therefore higher than those permitting growth in the original GW medium (median 80 CFU/mL; Wade and Graver, 2007). Although the rate of growth of N. gonorrhoeae in GW-An medium appears lower than in GW medium, there are differences not only in composition of the media but also in growth conditions: in our earlier report growth was in 50 mL GW medium in 5% CO2 agitated at 100 rpm on an orbital shaker. We believe there are intrinsic strain-dependent differences in growth rate despite provision of all the substrates necessary to satisfy the auxotrophic requirements of all strains tested. Comparing growth rates between time zero and time 20 h in the two studies (results not shown) we find that of the five slowest growing auxotypes in GW medium in CO2, four (including both AHU strains) are amongst the five slowest growing in the present study; similarly, two strains (auxotypes A and PAOU) were amongst the five most rapid growers in both studies. The ability to study N. gonorrhoeae growing anaerobically will advance our understanding of the pathogenicity of a bacterium of major public health significance. To date, anaerobic growth of N. gonorrhoeae has been achieved as a halo around sodium nitriteimpregnated discs (Knapp and Clark, 1984; Frangipane and Rest, 1992), on nitrite-containing media (Lissenden et al., 2000) or by the addition of pulses of nitrite to undefined liquid medium (Overton et al., 2006). ‘Oxyrase for Broth’ is a commercially-available biocatalytic oxygen-reducing agent containing Escherichia coli membrane fragments bearing enzymes capable of removing dissolved oxygen from liquid media within an hour of incubation at 37 °C and preventing subsequent oxygen ingress, thereby permitting the growth of strict anaerobes (Adler and Spadey, 1997). The 4.9 log increase in F. nucleatum counts over the initial 20 h period, and growth thereafter, confirm anaerobiosis; oxygen ingress from the minimal headspace was removed. The Oxyrase E. coli membrane fragments retain the ability to reduce nitrate (results not shown) and may provide adequate nitrite from the ferric nitrate present in commercially-available M199 medium to permit the growth demonstrated. In preliminary experiments additional nitrate or nitrite did
366
J.J. Wade, M.A. Graver / Journal of Microbiological Methods 79 (2009) 364–366
not increase growth rates (results not shown). The use of Oxyrase means that GW-An medium, unlike GW medium, is not strictly chemically defined. Further, the presence of E. coli membrane fragments and their associated enzymes may complicate the study of subcellular components of N. gonorrhoeae, though repeated centrifugation and washing of bacteria may reduce or remove this problem. It should be noted that, although lactate was omitted from GW-An medium, Oxyrase contains lactate as an enzyme substrate and this can be used by N. gonorrhoeae as an energy source. We are currently determining whether anaerobic growth of N. gonorrhoeae can be achieved when Oxyrase is separated from the basal medium by a dialysis membrane. The anaerobic growth of N. gonorrhoeae in the liquid medium described here – which is based on a cell culture medium – will simulate more closely the growth of this bacterium in anatomical relevant sites with a low partial pressure of oxygen, such as the female genital tract, than currently available solid or liquid bacteriological media. The medium described here offers a novel way to study growth of N. gonorrhoeae and its interactions with other bacteria under anaerobic conditions.
References Adler, H., Spadey, G., 1997. The use of microbial membranes to achieve anaerobiosis. J. Rapid Methods Automat. Microb. 5, 1–12.
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