British Journal of Venereal Diseases, 1978, 54, 386-393 Method for studying the role of indigenous cervical flora in colonisation by Neisseria gonorrhoeae M. E. McBRIDE, W. C. DUNCAN, AND J. M. KNOX From the Departments of Dermatology and Microbiology, Baylor College of Medicine, Texas Medical Center, Houston, Texas, USA SUMMARY A method for quantitating cervical flora has been evaluated statistically and used to study the bacterial flora of the cervix in 14 women sexually exposed to men with gonococcal ure- thritis. A comparison was made between those women who subsequently became colonised with Neisseria gonorrhoeae and those who did not to determine whether either total microbial popula- tions or the different species present could be related to colonisation by N. gonorrhoeae. Two control groups of healthy women, one of patients from a public clinic and the other of patients from a private practice, were studied in the same way. Normal flora isolates were tested in vitro for antagonism or synergism toward N. gonorrhoeae or both. Cervical flora was characterised in all patient groups by wide variations between individuals, both in type and numbers of organisms. No significant differences were found in total bacterial populations or in the number of species isolated from the cervix between patient groups. Populations of N. gonorrhoeae ranged from less than 10 bacteria to log,04'36. Only one normal flora isolate, a strain of Streptococcus viridans isolated from a woman exposed to but not infected by N. gonorrhoeae, demonstrated inhibition of growth towards N. gonorrhoeae. Introduction There has been increasing interest in the importance of the microbial flora of normal healthy individuals in maintaining a stable ecological balance which might protect against colonisation by pathogenic micro-organisms. This type of control can occur in different ways: as antagonism between microbial species (or bacterial interference); or by the avail- ability of the ecological space to invasion, which occurs in the absence or suppression of normal flora. Bacterial interference has been described in a variety of human ecosystems, and includes the organisms associated with dental plaque (Homberg and Halkinder, 1972), skin microflora (Selwyn, 1975), and the flora of the respiratory tract (Sprunt et al, 1971; Crowe et al, 1973), while suppression of normal flora as a result of antibiotic treatment has been known to result in colonisation by oppor- tunists. The complexity of the microflora of the female genital tract makes it difficult to assess its protective Address for reprints: Dr M. E. McBride, Department of Dermatology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030, USA Received for publication 16 March 1978 role against infection. The composition of vaginal and cervical flora has been a subject of interest since Doderlein's classical description of Lacto- bacillus in 1892. Hite et al (1947) reviewed some of the early literature while Galask et al (1976) re- evaluated the role of vaginal flora in disease. A comprehensive overview of the problems of studying the microflora of the female genital tract as an ecosystem was presented by Hurley et al (1974), and more recent definitive studies using quantitative techniques (Bartlett et al, 1977; Levison et al, 1977) have emphasised the complexity of the flora of the female genital tract. Although bacterial interference has been des- cribed between Neisseria gonorrhoeae and Staphylo- coccus epidermidis (Kraus and Ellison, 1974), Escherichia coli (Kraus et al, 1976), and Candida albicans (Hipp et al, 1974) studies indicating shifts in population groups related to infection have been few. deLouvois et al (1975) were able to correlate statistically the increase in isolation of Trichomonas vaginalis, Mycoplasma hominis, and Bacteroides species with a decrease of Lactobacillus. The observation that decreased frequency of isolation of Lactobacillus correlated with vaginitis and 386 copyright. on 1 October 2018 by guest. Protected by http://sti.bmj.com/ Br J Vener Dis: first published as 10.1136/sti.54.6.386 on 1 December 1978. Downloaded from
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British Journal of Venereal Diseases, 1978, 54, 386-393
Method for studying the role of indigenous cervicalflora in colonisation by Neisseria gonorrhoeaeM. E. McBRIDE, W. C. DUNCAN, AND J. M. KNOXFrom the Departments of Dermatology and Microbiology, Baylor College of Medicine,Texas Medical Center, Houston, Texas, USA
SUMMARY A method for quantitating cervical flora has been evaluated statistically and used tostudy the bacterial flora of the cervix in 14 women sexually exposed to men with gonococcal ure-thritis. A comparison was made between those women who subsequently became colonised withNeisseria gonorrhoeae and those who did not to determine whether either total microbial popula-tions or the different species present could be related to colonisation by N. gonorrhoeae. Twocontrol groups of healthy women, one of patients from a public clinic and the other of patientsfrom a private practice, were studied in the same way. Normal flora isolates were tested in vitro forantagonism or synergism toward N. gonorrhoeae or both. Cervical flora was characterised in allpatient groups by wide variations between individuals, both in type and numbers of organisms. Nosignificant differences were found in total bacterial populations or in the number of species isolatedfrom the cervix between patient groups. Populations of N. gonorrhoeae ranged from less than 10bacteria to log,04'36. Only one normal flora isolate, a strain of Streptococcus viridans isolatedfrom a woman exposed to but not infected by N. gonorrhoeae, demonstrated inhibition of growthtowards N. gonorrhoeae.
Introduction
There has been increasing interest in the importanceof the microbial flora of normal healthy individualsin maintaining a stable ecological balance whichmight protect against colonisation by pathogenicmicro-organisms. This type of control can occurin different ways: as antagonism between microbialspecies (or bacterial interference); or by the avail-ability of the ecological space to invasion, whichoccurs in the absence or suppression of normalflora. Bacterial interference has been described in avariety of human ecosystems, and includes theorganisms associated with dental plaque (Hombergand Halkinder, 1972), skin microflora (Selwyn,1975), and the flora of the respiratory tract (Spruntet al, 1971; Crowe et al, 1973), while suppression ofnormal flora as a result of antibiotic treatment hasbeen known to result in colonisation by oppor-tunists.The complexity of the microflora of the female
genital tract makes it difficult to assess its protective
Address for reprints: Dr M. E. McBride, Department ofDermatology,Baylor College of Medicine, Texas Medical Center, Houston, Texas77030, USAReceived for publication 16 March 1978
role against infection. The composition of vaginaland cervical flora has been a subject of interestsince Doderlein's classical description of Lacto-bacillus in 1892. Hite et al (1947) reviewed some ofthe early literature while Galask et al (1976) re-evaluated the role of vaginal flora in disease. Acomprehensive overview of the problems ofstudying the microflora of the female genital tractas an ecosystem was presented by Hurley et al(1974), and more recent definitive studies usingquantitative techniques (Bartlett et al, 1977;Levison et al, 1977) have emphasised the complexityof the flora of the female genital tract.Although bacterial interference has been des-
cribed between Neisseria gonorrhoeae and Staphylo-coccus epidermidis (Kraus and Ellison, 1974),Escherichia coli (Kraus et al, 1976), and Candidaalbicans (Hipp et al, 1974) studies indicating shiftsin population groups related to infection have beenfew. deLouvois et al (1975) were able to correlatestatistically the increase in isolation of Trichomonasvaginalis, Mycoplasma hominis, and Bacteroidesspecies with a decrease of Lactobacillus. Theobservation that decreased frequency of isolationof Lactobacillus correlated with vaginitis and
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Methodfor studying the role of indigenous cervicalflora
isolation of T. vaginalis was also recorded byGordon et al (1966).The purpose of this study has been to attempt to
identify individual differences in the microflora ofthe cervix in women exposed to, but not necessarilycolonised by, N. gonorrhoeae with the aim ofidentifying organisms which might exert a protectiveeffect. To do this a method for quantitation of thecervical flora has been devised and evaluatedstatistically. Two other groups of patients havebeen included in the study to provide a baseline fornormal variability: healthy women attending apublic premarital clinic and healthy women froma private gynaecological practice. Bacterial isolatesfrom each of these groups have been examined forinterference with N. gonorrhoeae in vitro.
Materials and methods
QUANTITATION OF CERVICAL FLORAStudy populationA total of 45 patients were studied. Of these, 14women attending the City of Houston PublicHealth Clinic were known recent sexual contactsof men with acute gonococcal urethritis; 11 werepresumably healthy women attending the premaritalclinic; and 20 were presumably healthy patientsfrom a private obstetrical practice reporting for aroutine cervical cytological smear. None of thepatients had taken antibiotics in the precedingthree weeks, and none had douched within 24hours.
Sampling methodsTwo Dacron-tipped swabs were used to obtainduplicate samples from the cervical os using aspeculum. The swabs were immediately placed inStuart's medium and transported to the laboratorywithin a maximum of three hours from the time thecultures were taken. A third cervical culture wasplated on Thayer-Martin medium for routineexamination by the Houston Public Health Labora-tory for N. gonorrhoeae. Identical sampling methodswere used for each group of women.
Culture methodsEach duplicate swab was placed in 1 0 ml of phos-phate-buffered saline (pH 7-2) and shaken on aVortex junior mixer for one minute. Smears of thissuspension were made for Gram staining. A 0 1 mlaliquot was inoculated directly on to a Casman'ssheep blood agar plate (Casman's Medium Base,Difco) and Rogosa's medium (Rogosa's SL Agar,Difco) and streaked. The 1-0 ml suspension wasfurther diluted using tenfold dilutions to 104.Each dilution was inoculated on to the surface of a
Casman's sheep blood agar plate using a calibrateddropping pipette and six drops per plate. Differentialmedia were used randomly to determine theirvalue; these included chocolate agar, Thayer-Martinmedium, tomato juice agar (Difco), and Rogosa'sagar, which were found to be the most useful.Casman's sheep blood agar was superior to choco-late agar for the isolation of fastidious organisms,and Rogosa's agar was found to the most useful forgrowth of Lactobacillus. Other selective media didnot provide additional information. In certainpatients duplicate plates of Casman's sheep bloodagar were inoculated for growth in an anaerobic jar(Gaspak). Rogosa's agar was incubated anaerobic-ally using the Gaspak system at 37°C. Aerobicincubation supported only a very light growth oforganisms such as E. coli, Micrococcus, et cetera.Growth was visible at 48 hours, but further colonialdifferentiation could be observed at 72 hours,particularly the development of haemolysis. Theappropriate culture dilution (usually 103 or 104)was chosen with the aid of a stereoscopic micro-scope; it was then possible to do a differential countbased on colonial morphology. Species present inlow numbers were counted from the plate streakedwith 0-1 ml inoculum. Identification and charac-terisation were carried out using methods of Cowanand Steele (1975).At the Public Health Laboratory suspected
colonies from cervical cultures were Gram-stained,and the oxidase reaction was determined. Wherethis reaction was positive and typical Gram-negative diplococci were present the organism wasconsidered to be N. gonorrhoeae.
Statistical methodsA paired t statistic was used to compare totalcounts in duplicate specimens. Multiple regressionevaluated by an F statistic was used to determinethe correlation between the populations of speciesisolated from the duplicate samples after correctingfor the total counts of the duplicates.
BACTERIAL INTERFERENCE
Screening method 1A suspension was made of a fresh clinical isolate ofN. gonorrhoeae in phosphate-buffered saline (pH7-2) and diluted from 101 to 104. An 0-1 ml aliquotof each dilution was plated on the surface ofCasman's sheep blood agar plates. Isolates ofnormal flora were spotted on the surface of theN. gonorrhoeae lawns. The plates were incubatedin a CO2 jar at 37°C and examined at 24 and 48hours.
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Screening method 2Lawns were prepared on Casman's sheep bloodagar with isolates of normal flora organisms.Dilutions of an overnight culture of a fresh isolateof N. gonorrhoeae were spotted on to the lawns bymeans of a calibrated pipette delivering a constantinoculum. Control plates of Casman's sheep bloodwithout normal flora were inoculated in the sameway. Plates were incubated in a CO2 jar at 37°C
saline and inoculated on to Casman's sheep bloodagar and Thayer-Martin medium by means of acalibrated dropping pipette. These plates wereincubated in a CO2 jar at 371C for 48 hours, atwhich time a colony count was done and a growthcurve plotted.
Results
and examined at 24 and 48 hours for inhibition or Since the cervical specimen was suspended insynergism of growth of N. gonorrhoeae or both. diluent before plating, organisms present in low
numbers were not cultured. When 0-1 ml ofGrowth curves of mixedpopulations inoculum suspension was used on a streak plateNormal flora organisms giving some indication of common species associated with genital tractsynergism or antagonism were chosen for growth flora-for example, E. coli and other entericin mixed cultures. Preliminary experiments were organisms, Staph, epidermidis, Micrococcus species,done to achieve an equivalent concentration of and diptheroids-could be readily enumerated.each species of bacteria as inoculum. Screw-topped These organisms represent a relatively low propor-test tubes containing 50 ml of Casman's broth tion of the total flora and seem to be more of acontaining 5% sheep's blood were inoculated at 'contaminant' flora since further dilutions of the371C for five days. Control pure cultures of each sample yielded high populations of more fastidiousof the species were inoculated and incubated under organisms. Figures 1 and 2 show growth fromthe same conditions. Aliquots were removed from inoculum delivered from a calibrated droppingthese cultures daily, diluted in phosphate-bufferedApipette of 102 (x 1 3 magnification) and 104 (x 2 5
Fig. 1 Casman's sheep blood agar plate inoculated with 102 dilution of cervical specimen ( x 1 -3 magnification)
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Method for studying the role of indigenous cervical flora
magnification) dilution of a typical specimen.Different colony types are evident, and a differentialcount can be done on the basis of colonial mor-phology. The flora was predominantly CO2 depen-dent and facultatively anaerobic, with the exceptionof Bacteroides, some Lactobacillus species, Veillonellaspecies, and a group of Gram-negative cocci whichwere strictly anaerobic. Identifiable genera foundin high populations included a variety of strepto-cocci, Eikenella, Haemophilus vaginalis, and otherHaemophilus species. Many isolates did not readilyconform to identification schemes.
Evaluation of quantitative methodFor statistical evaluation of the methods the resultsfrom all patient groups were pooled. Table 1shows the range of microbial populations in duplicatesamples A and B taken from the cervix. The cervicalflora was found to be very variable between indi-viduals both in total populations and in differentspecies present, but duplicate cultures of the cervixtaken in a uniform manner were found to contain
statistically comparable microbial populations. Thedistribution of the total populations from duplicatecultures is shown in Figure 3. There is a normaldistribution of populations, the majority of patientshaving populations ranging between log,04 tolog107 colony-forming units (cfu) per ml per sampleinclusive.
Comparison of populations of different speciesisolated from duplicate cervical culturesA total of 115 isolates was obtained from the 45patients, and only three of these did not appear inthe duplicate cultures. There were 12 patients with
Table I Comparison of microbial populations ofduplicate cultures of the cervix
Fig. 3 Distribution of total populations from duplicatecultures from the cervical os
pure cultures of a single organism, and the largestnumber of isolates per culture was seven. Thepopulations of the individual species in duplicatecultures were compared by means of the methodof multiple regression. After regressing the indi-vidual species of culture sample B from the totalpopulations of sample A and duplicate sample B,there remained a large regression coefficient (R)for the individual species (B). R square for the totalfor sample A plus the total for sample B was 0-32,while for total populations for sample A plus totalpopulations for sample B plus the values for theparticular species in sample A was 0-86. TheF statistic for an individual species was 397-9 on1 with 106 degrees of freedom, which is significantat the 0-001 level. Thus the populations of speciesisolated from duplicate cultures A and B werehighly correlated even correcting for total counts.
Comparison of bacterial populations in differentpatient groupsThe total bacterial populations were calculated foreach patient group to determine whether individualswith lower populations of indigenous microflorawere more susceptible to colonisation by N. gonor-rhoeae. Similarly, the importance of a more variedflora, as demonstrated by the number of differentspecies isolated, was examined. Bacterial popula-tions of the cervix of the three patient groupsstudied (healthy practice patients; healthy clinicpatients; and asymptomatic sexual contacts ofN. gonorrhoeae) are shown in Table 2. The rangeand mean values of the total population in thespecimen were comparable between the threegroups. The results from the 14 contacts of N.gonorrhoeae were broken down further into thosewith positive results from cultures for N. gonorrhoeae
and those with negative results. The presence orabsence of N. gonorrhoeae in the cultures wasdetermined independently at the Public HealthLaboratory and correlated with the results of thisstudy; half the patients were found to harbourN. gonorrhoeae. The results are shown in Table 3.Although the microbial populations per sample
Table 2 Comparison of bacterial populations of thecervix in three patient groups
Microbial populations(logiocfu/specimen) No. of isolates
were lower in patients colonised by N. gonorrhoeae,with a mean of 4-99 log,ocfu per sample comparedwith 5-94 log&.cfu per sample from those patientswho were not colonised, the difference was notstatistically significant (t= 1 -276). In addition therewas no difference in the number of isolates obtainedfrom each sample between the two groups. A lowerpopulation of indigenous flora, therefore, did notappear to be a predisposing factor to colonisationby N. gonorrhoeae.The relative populations of N. gonorrhoeae to
the total bacterial population of the specimen isshown in Table 4. Although three of the sevenpatients had relatively high populations of N.gonorrhoeae in their cervical cultures, none hadpure cultures. Four patients had a population ofN. gonorrhoeae of less than 10 bacteria. The high orlow population of N. gonorrhoeae did not seem tocorrelate either directly or indirectly with high orlow total populations. Similarly, the number ofother species present appeared to bear no relation-ship to colonisation by N. gonorrhoeae.
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In an attempt to identify qualitative differences inflora between patient groups it was obvious thatthe differences between individuals were so greatwithin each group that it would be very difficult toassess their relative importance. Figure 4 shows theflora of two healthy patients from the privatepractice group demonstrating the differences inboth total populations and numbers of speciespresent. These qualitative and quantitative differ-ences were noted in each patient population studied.Lactobacillus was the only genus which showedrecognisable differences between the three patientgroups studied. The populations and incidence ofisolation of Lactobacillus species from individualsexposed to N. gonorrhoeae in normal clinic patientsand those from a private practice are shown inTable 5. Only one patient exposed to N. gonorrhoeae(but not infected) and two of the healthy clinicpatients were colonised with Lactobacillus comparedwith 11 of the 20 private patients. Populations ofLactobacillus overall ranged from log,03-9 to 6-8cfu per specimen, representing 17-9% of the totalbacteria present in one specimen to 100% in others.
Table 5 Comparison ofpopulations of Lactobacillusspecies in three patient groups
Lactobacillus species
Patient group Population % of total bacterial(logiocfu/ml specimen) population
Fig. 4 Comparison of cervicalflora in two healthycontrols
Bacterial interferenceThe majority of the isolates obtained from eachpatient group were screened to demonstrate inhi-bitory or stimulatory activity towards N. gonorrhoeaein vitro. Only one isolate, a strain of Streptococcusviridans (obtained from a contact of N. gonorrhoeae),demonstrated inhibition towards N. gonorrhoeae.This patient had a negative culture result for N.gonorrhoeae. The growth of N. gonorrhoeae andthe isolate of Str. viridans was followed in mixedliquid culture medium for four days, and the resultsare shown in Figure 5a. The growth of Str. viridansshowed very little difference when grown in pureor mixed culture. The growth of N. gonorrhoeae inpure culture reached a high level and this wasmaintained for five days. In mixed culture, however,this organism did not achieve a high populationlevel before showing a steady decline until noviable organisms were detected at four days.The growth of N. gonorrhoeae in mixed culture
with an organism isolated from normal flora froma patient colonised with N. gonorrhoeae is shown inFigure Sb. Growth of the normal flora isolate isnot affected by N. gonorrhoeae. The growth ofN. gonorrhoeae, while inhibited during the earlystages of growth, within four days graduallyreached populations only slightly lower thanN. gonorrhoeae grown in pure culture. This isolateof normal flora is characteristic of cervical flora.It is a fastidious CO2-dependent 3-haemolyticpinpoint colony showing Gram-variable-stainingand pleomorphic cocco-bacillary types, oxidase- andcatalase-negative and penicillin-sensitive.Only one normal flora isolate, cultured from a
private patient, showed inhibition by N. gonorrhoeaeduring the screening procedure.
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Fig. 5 Growth curves of mixed cultures ofN. gonorrhoeae and normal flora isolates(a) 0 0 N. gonorrhoeae in pure culture; o- - -o
N. gonorrhoeae in mixed culture; A A-Streptococcus viridans in pure culture; A- - -A
Streptococcus viridans in mixed culture
Discussion
In an attempt to relate the role of normal flora ofthe cervix to colonisation by N. gonorrhoeae thereliability of a method of quantitation is essential.While the method described here can be consideredonly semi-quantitative, in as much as the samplesize was not quantitated, the duplicate samplingprocedure showed that it is possible-by uniformsampling-to obtain reliable quantitative results.Although the total microbial populations aresemi-quantitative, the relative populations of differ-ent species are not in as much as they can be relatedto a fixed volume of sample material. When someof the differences in technique are taken intoconsideration the findings presented here con-cerning population levels of cervical flora are
basically comparable to those described by Bartlettet al (1977).The protective role of indigenous flora against
colonisation by N. gonorrhoeae is not obviouseither by the population levels utilising the eco-logical space or by the presence of any one parti-cular species. Individuals with high populations ofnormal flora were colonised by N. gonorrhoeaewhile some exposed individuals with low cervicalpopulations were not. In addition it was not possibleto identify a characteristic flora from patientsexposed to, but not infected by, N. gonorrhoeae.
(b) 0 N. gonorrhoeae in pure culture; o- -o N
gonorrhoeae in mixed culture; N * normalflora isolate in pure culture; o----o normalflora isolate in mixed culture
Although a number of different species of strepto-coccus were isolated, in only one patient wasStr. viridans present and this showed inhibitiontowards growth of N. gonorrhoeae. This patienthad contact with N. gonorrhoeae but was notinfected. It would seem, therefore, that protectionagainst infection by normal flora species wouldbe a fortuitous event. Str. viridans has been knownto demonstrate bacterial interference against groupA Streptococcus pyogenes in the respiratory tract,where it occurs with a higher frequency of isolationand in high populations (Sprunt et al, 1971; Croweet al, 1933). While the results of screening isolatesfor bacterial interference are largely negative, thismay be a result of the in-vitro screening techniqueand may not reflect the in-vivo environment.The individual variability, both quantitative and
qualitative, in indigenous cervical flora emphasisesthe lack of information on the factors effectingnormal flora. Thus, the importance of studyingpopulation fluctuations in individuals is obvious.Although a quantitative technique has served toelucidate the relative importance of various speciescomprising normal flora, our knowledge is stillhampered by the lack of definition of many organ-isms present, many of which are extremely fasti-dious and do not conform to classification schemes.The wide fluctuations in N. gonorrhoeae
populations in women exposed to infection indicates
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Method for studying the role of indigenous cervical flora
some sort of innate protection, even though one ofthe most significant factors in the initiation ofinfection, the infectious dose, is unknown. Thenature of this protection may be related to a multi-plicity of factors, including the chemical environ-ment and immunological factors. The protectiverole of the indigenous flora against N. gonorrhoeaecannot be dismissed by this limited study, but amethod is presented for an area that should bepursued.
The technical assistance of Adelia McBride isgratefully acknowledged.
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