EPIDEMIOLOGICAL SURVEY OF PENICILLINASE


PRODUCING NEISSERIA GONORRHOEAE (PPNG)

IN ZIMBABWE

by

CLAUDE TAURAI SABETA

A thesis submitted in partial fulfilment of 
the requirements for the degree of 
Master of Philosophy (M.Phil).

*36005005720*

University of Zimbabwe Library

Department of Biological Sciences

Faculty of Science

University of Zimbabwe

JUNE 1991


ii

'•W

ACKNOWLEDGEMENTS

The author wishes to take this opportunity to express his appreciation 

to his research advisor; Dr J. M. Gopo for his support throughout the study;

without him it could have been very difficult to collect specimens around the 
country and the following members of his advisory committee: Dr S. Tswana 
and Dr S. B. Feresu. The author also expresses his sincere appreciation to Dr

S. B. Feresu and Professor P. R. Mason for their help during preparation of 

the manuscript. Special thanks go to the Department of Medical Microbiology 
for allowing the author to use its CO2 incubation facilities and Professor P. 
R. Mason and Mr L. Gwanzura for providing the author with some bacterial 

strains and with advice in culturing and identification of the organisms. Thanks

go to Mr F. Sibindi and Mr T. Mahachi for the technical help rendered especially 
with the photography of gels.

The study would not have been possible if it were not for the contribution 
of the following persons: Dr L. Mbengeranwa (City Help Department - Harare),

Dr Nyathi (City Health Department - Bulawayo), Mr V. Ncube and Mrs Hlongwani 

(Mpilo Hospital - Bulawayo). Mr Khosa (Khami Clinic - Bulawayo), Mr Foxton 

(Mutare City Health), Mr Chigumira (Kadoma Hospital), Mr Ahmed (Chinhoyi 
Hospital), Dr Coennen (Zvishavane Hospital), Mr Munyoro (Shabanie Hospital),

Dr Mapanda (Torwood Hospital), Mr Gorogodo (Kwe Kwe Hospital), Mrs Shumba 

(Gutu Rural Hospital), Dr Bango (Ministry of Health), Sister Paraiwa (Mbare

GUC Centre) and Mr Ndirayi (Mutare General Hospital).

The research study was supported by a University of Zimbabwe Research

Grant Vote No. 2.9999-10-3044.


iii

CONTENTS

CHAPTER Page

LIST OF TABLES ...............................................................................................v

LIST OF FIGURES .............................................................................................vi

ABSTRACT .......................................................................................................-vii

1 INTRODUCTION AND LITERATURE REVIEW ..................................... 1

2. MATERIALS AND METHODS ........................................................................ 20


2.1 Specimen Collection ................................................................ 20


2.2 Isolation of N. gonorrhoeae .........................................................22


2.3 Identification tests .................................................................... 22


2.3.1 Gram's Stain ................................................................. 22


2.3.2 Oxidase test ............................................................. 23


2.3.3 Acid production from carbohydrates .................................... 23


2.3.4 Antibiotic sensitivity tests ..................................................... 23


2.3.5 Beta-lactamase activity .......................................................... 24


2.4 Reference Strains ...................................................................... 24


2.5 Isolation of plasmid DNA ......................................................... 24


2.6 Preparation of gels ............................................................... 26


2.7 Electrophoresis ........................................................................... 26





iv

3. RESULTS




3.1 Isolation of N. gonorrhoeae ....................................................... 28


3.2 Antibiotic sensitivity tests ........................................................ 29


3.3 Comparison of beta-lactamase activity and penicillin G




resistance ..................................................................................... 30

3.4 Plasmid DNA analysis ................................................................ 31


3.5 Various plasmid Combinations ....................................................32


4. DISCUSSION .............................................................................................. 38




REFERENCES ............................................................................................ 43

APPENDICES ............................................................................................ 53


V

TABLES

Page

Table 1. Overall isolation rates for the different provinces for 
the period July 1987 - July 1989 ................................................... 28

Table 2. Results obtained for antibiotic resistances for the regimens used 
in the treatment of gonorrhoea .................................................... 29

Table 3. Comparison of numbers of beta-lactamase positive 
strains and those that were resistant to penicillin G .................. 30

Table 4. Plasmids detected in PPNG strains by agarose gel 
electrophoresis ............................................................................. 31

Table 5. Plasmid combinations obtained for all the provinces ............. 32

1


vi

FIGURES

Page

1. Inactivation of penicillin antibiotics by beta-lactamase ................... 14


2. Map showing areas where sample collection was done ............................. 21


3. Electrophoretic pattern of plasmid extracts from N. gonorrhoeae...... 34


4. Electrophoretic pattern of plasmid extracts from N. gonorrhoeae ....... 35


5. Electrophoretic pattern of plasmid extracts from N. gonorrhoeae...... 36


6. Electrophoretic pattern of plasmid extracts from N. gonorrhoeae ....... 37





ABSTRACT

The study involved an investigation into the plasmid types which confer resistance 
to penicillin in N. gonorrhoeae in Zimbabwe, and to determine the most occurring 
plasmid associated with PPNG.

Specimens were collected from patients presenting with urethral discharge 
at chosen hospitals and clinics in Zimbabwe. These were then cultured on selective 
Thayer-Martin (TM) media. Presumptive gonococci were subjected to Gram 
stain, oxidase and carbohydrate fermentation tests. Beta-lactamase activity 
of the isolates was assessed by using commercial intralactam strips. Antibiotic 
sensitivity tests were done using discs incorporated with penicillin G, augmentin, 
erythromycin and tetracycline. Plasmid DNA was isolated using an alkaline 
selective denaturation technique that destroys chromosomal DNA whilst retaining 
small covalently closed DNA molecules. The DNA so obtained was checked 
for size, homogeneity and purity using agarose gel electrophoresis. The plasmid 
profiles were used to ascertain the most prevalent plasmid type which conferred 
resistance to penicillin in N. gonorrhoeae isolates.

Four hundred and thirty six isolates of N. gonorrhoeae were isolated from the 
865 specimens collected. Of these isolates 147 (34%) were R plasmid bearing. 
All beta-lactamase producing strains were resistant to penicillin G. In addition, 
seven non-beta-lactamase producing strains were also resistant to penicillin 
G, possibly indicating chromosomal resistance. Plasmid analyses indicated 
that of the 147 strains tested, 144 (98%) harboured the 2.6 Md cryptic plasmid, 
56 (38%) the 3.2 Md plasmid, 66 (45%) the 4.5 Md plasmid. The 24.5 Md transfer 
plasmid existed in 61 (41%) of the strains. Eight isolates harboured both the 
3.2 and 4.5 Md plasmids.

The study demonstrated that resistance to penicillin in N. gonorrhoeae in Zimbabwe 
is equally confered by both the 3.2 Md and 4.5 Md plasmids.


1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Sexually transmitted diseases (STDs) are a major health problem worldwide. 
They are an important cause of morbidity in every country of the world. Their 
impact is seen not only in men and women experiencing immediate symptoms, 
but also in those who suffer long term secondary complications and in infants 
of infected women (1). Complications include ectopic pregnancy in young females, 
genital organ damage such as pelvic inflammatory disease (PID) in females, 
epididymoorchitis in males and ophthalmia neonatorum and congenital infection 
in babies (2).

Actual numbers of infection are unreliable from conventional statistics 
because in large parts of the world, as many as 90% of STDs go unreported. 
However, of those reported, the most prevalent particularly in Africa and the 
Third World are gonorrhoea, syphilis and chancroid. In Zimbabwe, STDs are 
highly prevalent diseases and contribute significantly to the work load and economics 
of curative medicine. In 1984, 133 676 patients were treated for STDs at clinics 
in Harare alone. The majority of cases (63%) were adult males, followed by 
adult females (36%), children under 15 years of age making up the remainder 
(1%) (3). In 1987, the Ministry of Health reported 4000 STD cases in just one 
province, Masvingo. The figures for all STDs throughout Zimbabwe for 1988 
were of the order 900 000 (4). These figures demonstrate that STDs in Zimbabwe 
are highly prevalent and form a major contribution to morbidity statistics. 
Amongst the most common STDs is gonorrhoea, and the organism causing this 
disease is Neisseria gonorrhoeae. The penicillin resistant strains of N. gonorrhoeae, 
PPNG, are the subject of this thesis.


2

Gonorrhoea

Gonorrhoea is a communicable disease, transmitted between individuals 
mainly by sexual contact, and caused by Neisseria gonorrhoeae. The sites that 
are normally infected are the distal urethra in males and the cervix in females. 
In both sexes, infection of other mucous membranes such as the rectum and 
pharynx may occur, as well as infection of the conjunctiva in babies. The incubation 
period is normally 24 - 48 hours but is variable. Following this period, infection 
usually stimulates discharge from the distal urethra and pain during urination 
(5). In males, the most common manifestation of infection is dysuria with a 
purulent discharge. Infection in females is often asymptomatic. The incidence 
of asymptomatic gonorrhoea in males is between 3 to 10%, whereas in women 
it is estimated to be 40% or higher (5).

Neisseria gonorrhoeae is a Gram negative, non-motile^ non-spore forming 
coccus which is usually seen in pairs, but occasionally in tetrads or clusters. 
The bacterium grows optimally at 27 - 35°C but not at 22°,C. It is aerobic, 
produces oxidase and degrades glucose but not maltose or sucrose. In addition, 
gonococci are sensitive to toxic fatty acids and trace metals and as a result 
it is difficult to culture gonococci. Whole blood is usually required to supplement 
growth media.

Neisseria gonorrhoeae has proteinaceous appendages on its cell wall that 
have been implicated in bacterial adherence, regarded as a prerequisite for 
colonisation of the human host (6). Much interest has therefore been focussed 
on proteins in the gonococcal outer membrance. This membrane is composed 
mainly of lipopolysaccharides in which the proteins are embedded. The major 


3

proteins have been termed Pl and P2. The predominant protein species is Pl 
which is thought to function as a porin (7). Porins are sites that allow entry 
or exit and exclusion of many substances, including antibiotics, and may therefore 
play an important role in antibiotic resistance. Gonococci that do not possess 
P2 proteins in their outer membrance are thought to be more virulent and invasive 
(8). Under iron starvation, gonococci express markedly increased amounts of 
the outer membrane proteins (9).

Resistance to antimicrobials

Since gonorrhoea is a common infection and can recur frequently, its 
ideal treatment should be effectice, cheap, preferably given in a single dose 
and be free of side effects. These requirements were initially met by sulfanilamide, 
one of the sulphonamide family of antibiotics in the 1930's (10). However, resistance 
to these antibiotics emerged rapidly, probably because of selective pressure 
caused by their extensive use. Fortunately, at the same time penicillin began 
to be widely available, and was found to be effective in the treatment of gonococcal 
infections. Slow development of penicillin resistance in gonococci was detected 
from the mid-1950's, and by the 1970's the curative dose had to be increased 
thirty-fold from 150 000 units to 4 800 000 units of Penicillin G. This was given 
with Probenecid, a drug which inhibits renal tubular secretion of Penicillin G 
and thus maintain higher and more prolonged tissue levels of the antibiotic, 
thereby increasing the time the microorganism is exposed to effective levels 
of the drug (11).


4

In 1976 there emerged strains of gonococci producing penicillinase and 
treatment regimens had to change because these were totally resistant to penicillin. 
Regimens were adopted that were based on the susceptibilities of gonococci 
in different localities (5). The Group A regimens (amoxicillin, ampicillin, benzylpenicillin, 
procaine penicillin, doxycycline and tetracycline) were recommended in areas 
where penicillinase-producing N. gonorrhoeae (PPNG) comprised less than 1% 
of gonococcal isolates. Group B regimens (spectinomycin, ceftriaxone, cefotaxime 
and cefoxitin) were recommended in areas where chromosomal resistance had 
reduced the efficacy of Group A antimicrobial agents to below 9596 of infections 
and in areas where PPNG exceed 596 of isolates. Group C regimens (kanamycin, 
thiamphenicol, trimethoprim/sulphamethoxazole) were recommended for areas 
where PPNG were prevalent. They are widely used and considered more effective 
than spectinomycin and third generation cephalosporins (5).

Chromosomal antimicrobial resistance

N. gonorrhoeae has shown resistance to antibiotics by basically two mechanisms: 
by mutation of chromosomal loci and by the plasmid mediated production of 
enzymes that destroy beta-lactams. Mutations on chromosomal loci have been 
shown to modify the target site for the antibiotic in such a way that the site 
no longer accommodates the drug (11).

Although resistance by N. gonorrhoeae to penicillin has recently been

attributed to production of penicillinases, there has been a long history of resistance 
or at least reduced susceptibility by non-penicillinase producing strains. High 
level resistance may be shown by such strains, for example that isolated by


5

Faruki and Sparling (12) in the United Kindgom. It was demonstrated that this 
strain of N. gonorrhoeae had mutations at the genetic loci pen A, pen B and 
tem. The strains did not contain any of the plasmids coding for penicillinase 
production, and resistance must therefore have been mediated through changes 
in penicillin binding proteins (PBP) in the cell wall. The strains were termed 
chromosomally mediated resistant gonococci (CMRG) (13).

Another example of chromosomal resistance was demonstrated by Spratt

(14,15) who isolated strains that did not express high levels of resistance to 
penicillin but had mutated genes in four loci which resulted in the production

% ¦

of altered forms of PBP. These proteins had decreased affinity for penicillin

and as a result the rate of penetration of the antibiotic through the outer membrane 
was greatly reduced. Since binding to PBP is essential for the antimicrobial 
activity of penicillin these strains showed decreased susceptibility to this antibiotic.. 
This form of resistance, mediated through Changes in PBP structure, can be 
overcome by increasing the dose of penicillin. Much greater levels of resistance 
that could not be overcome by high dose regimens, were found in strains with 
plasmids coding for beta-lactamase enzymes.

Plasmid Antimicrobial Resistance

Plasmids

Plasmids are strands of extrachromosomal supercoiled circular DNA that 

are found in most bacterial species as well as in some species of eukaryotes.


6

Plasmids are non-essential for the growth of normal cells of the host species 
and can either be lost or gained without any major effect on the organism (16). 
Many plasmids however contain genes that may become essential for the survival 
of bacteria in certain environments. For example, R plasmids carry genes that 
confer resistance to antibiotics. In nature, a cell containing such a plasmid 
can survive in the presence of natural antibiotics produced by co-existing fungi 
(17).

Although plasmids are dependent to a large extent on the metabolic activity 
of the host cell, they can replicate independently of the chromosome of the 
cell. Each type of plasmid has its own genes for regulating the number of plasmid 
copies per cell. This number may range from 1 or 2 for the stringent or low 
copy number plasmids, to 10 - 100 for the relaxed or high copy number plasmids.

Plasmids confer a variety of phenotypic traits on bacteria (18). These 
include bacteriocin production, resistance to heavy metals and drugs, induction 
of plant tumours, sugar fermentation, degradation of aromatic compounds, 
haemolysin and hydrogen sulphide production. Host controlled restriction and 
modification as well as production of virulence factors can also be coded for 
by plasmids. There are three main types of plasmid which have been studied, 
the fertility (F), resistance (R) and the colicin (Col) plasmids. Colicin plasmids 
play no role in the development of resistance to antimicrobials, and so are beyond 
the scope of this thesis. Some plasmids do not confer any discernible phenotypic 
characteristics on an organisms and so are called cryptic plasmids. Plasmids 
may be classified into two major types, namely infectious and non-infectious 
plasmids (16). Infectious or self-transmissible plasmids can transfer themselves 
from one bacterium to another by either transformation or conjugation.


7

Transformation is a process of intercellular transfer of information in 
which a fraction of the donor cells total DNA can penetrate into a related bacterial 
cell. The actual mechanism of transfer is unclear, but probably involves movement 
of DNA across cell membranes while the latter are in the state of dynamic 
activity, for example during late log phase of active growth. Once in the cell, 
the donor DNA can be incorporated through the process of recombination into 
the recipient's DNA. If the newly integrated sequence derived from the donor 
differs in part from the nucleotide sequence of the recipients, new information 
for the characters controlled by this DNA region will be provided for the transformed 
cell.

The DNA involved in transformation can only be taken up by the recipient 
if the recipient is in a 'competent' state. Even in species, in which transformation 
has been demonstrated, different strains differ in their ability to be transformed. 
Neisseria gonorrhoeae are unusual in the ease in which they can be genetically 
transformed with naked transforming DNA (8). Competence for transformation 
is much greater in piliated (Pil+) colonial forms of N. gonorrhoeae than in their 
isogenic non-piliated (Pil") colonial variants and competence may be lost in 
vitro during transition from Pil+ to Pil-. While this may imply that pili are 
directly responsible for the ability to take up transforming DNA, perhaps facilitating 
transformation by acting as receptors or by creating channels through the outer 
membrane, there is no direct evidence to suggest a role for pili in transformation 
itself (19).

Neisseria gonorrhoeae are presumed to be highly competent in vivo. Circular 
plasmid DNA is more effectively taken up by recipient cells than gonococcal 
chromosomal DNA released by autolysis. Transformation may thus be a principal 
means of exchanging genes between gonococci and other Neisseria. Transforming 
DNA is taken up by gonococci in its double-stranded form and is maintained 


8

in this form until just before recombination with the recipient chromosome.

Some of the properties that can interfere with transformation are the secretion 
of DNase (which can inactivate transforming DNA) and the unusual cell surface 
barriers presented by dense mucoid capsular materials (which interfere with 
the penetration of DNA into the cell). The presence of nucleases poses an important 
limitation on the survival of naked DNA thus reducing the importance of transformation 
as a means of gene transfer.

Plasmids can also be transferred from one bacterial cell to others through 
bacterial conjugation. The process requires direct cellular contact between 
a donor (male) bacterium and a recipient (female) bacterium. Following such 
a contact and formation of a connecting bridge, a segment of the ’male’ chromosome 
may be transferred into the ’female’ where it undergoes recombination with 
a corresponding segment of the recipient. Much larger fragments of the donor’s 
genome are tranferred in bacterial conjugation than in transformation.

F factors

The F plasmid is normally a small covalently-closed circular molecule

of DNA that is approximately 2% the size of the chromosome of E. coli, the 
organism in which such plasmids were first demonstrated. F plasmids can either 
exist autonomously or can be integrated into the bacterial chromosome. The 
importance of F plasmids lies in their ability to enhance DNA transmission 
between bacterial cells. Cells that carry the F plasmid are termed F+ cells 
and those that do not carry the F plasmid are termed F~. Sex pili can be demonstrated 
during the exponential growth phase of cells that carry the F plasmid. These 
long, thin proteinaceous appendages are believed to be encoded by the F plasmid.


9

Cell-cell interaction between F+ donor and F- recipient occurs at random 
resulting in the formation of a mating pair. It is unclear whether the DNA 
is tranferred through the sex pilus itself or whether some sort of additional 
bridge is required. Transfer of DNA is unidirectional, that is, from F+ to F*. 
A single linear strand of F enters the recipient cell leaving the complementary 
strand in the donor. The single strands are immediately used as templates for 
synthesis of double stranded DNA. The recipient cell allows the single strand 
of foreign DNA to replicate and circularise in the presence of its nucleases. 
The recipient cell harbouring the transferred plasmid becomes a further vehicle 
for F transmission (20). F plasmids also mediate exchange of DNA sequences 
other than those of themselves. This process of gene transfer is sometimes 
referred to as F-duction. The initial step of F-duction involves integration 
of the plasmid into the donor's DNA, and bacteria that harbour F in this manner 
are said to be Ilfr (high frequency of recombination of donor markers). Integration 
of F into the bacterial chromosome occurs at particular sites at a frequency 
of 10‘5 to IO"? per generation.

Since the transfer system is still active in the Hfr system, the donor's 
DNA is mobilised because F is now part of the bacterial chromosome. The 
mechanism of mobilisation of the bacterial chromosome appears similar to 
that of F. Entry of the entire chromosome of the donor cells occurs very rarely 
since random shear of the linear single-stranded donor DNA terminates the 
process spontaneously. In practice only part of the F plasmid and of the donor 
DNA is transferred. The F plasmid is also able to transfer smaller plasmids 
from one bacterial cell to the next. Thus the F plasmid is a conjugative plasmid.


10

Conjugative plasmids in N. gonorrhoeae

A high proportion of penicillin resistant (Pcr) gonoccoci and as many as 
22‘b of unselected gonococci possess a 24.5 Md plasmid that has conjugative 
properties. This conjugative plasmid is able to mobilise or transfer smaller 
plasmids that carry resistant gene markers from one bacterial cell to the next 
(21).

R Plasmids

The genetic element responsible for transfer of drug resistance in bacteria 
is often a plasmid and is termed an R factor. It has been assumed that the 
origin and evolution of R factors is equivalent to that of F factors. A transfer 
factor is proposed to have integrated into the bacterial chromosome near a 
chromosomal gene mutated to drug resistance. On release of the transfer factor 
from the chromosome, the adjacent bacterial region was incorporated as part 
of the newly created R factor. Such an R factor could build up resistance to 
a number of drugs in this manner. Thus an R factor consists of an infectious 
unit plus resistance markers called r-determinants. The resistance transfer 
factor (RTF) region enables the plasmid to be transmitted to other bacteria 
by conjugation and so genes in the RTF region closely resemble those in F factors 
(22). Small R factor plasmids, devoid of the RTF regions are transmissible 
only with the aid of a conjugal plasmid.


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Gonococcal Plasmids

A number of plasmids have been described from N. gonorrhoeae. These

include a 2.6 Md cryptic plasmid as well as the 3.2 and 4.4/4.5 Md plasmid (different 
workers having given slightly different molecular weights) which confer resistance 
to penicillin and similar beta-lactam antibiotics. In this thesis, the large R- 
plasmid will be referred to as 4.5 Md plasmid. Other gonococcal plasmids include 
a 24.5 Md conjugative plasmid and a 25.2 Md plasmid associated with tetracycline 
resistance. These plasmids evolved either naturally within N. gonorrhoeae or 
were exogenous in origin and were transferred to N. gonorrhoeae by either transformation 
or conjugation. The 2.6 Md and 24.5 Md plasmids appear to be of endogenous 
origin based on studies of their G:C ratio, hybridisation techniques and stability.

The other plasmids are thought to be of exogenous origin. The presence of 
each plasmid in gonococci is highly variable (23).

2.6 Md plasmid




The 2.6 Md plasmid was the first plasmid to be discovered in the gonococcus

and has been detected by various methods including electron microscopy and 
density gradient centrifugation. This small plasmid comprises 4200 base pairs 
with a G:C content of 5096 (23). Its complete nucleotide sequence has been 
determined. The plasmid has been found in almost all gonococcal isolates with 
the exception of a few which belong to a specific auxotype. The 2.6 Md plasmid 


12

is cryptic and is not correlated with the virulence of the gonococcal strains, 
pili production, nor with the gonococcal outer membrane. Large segments of 
the 2.6 Md plasmid can be integrated into the chromosome in both plasmid bearing 
and plasmid-free strains (23).

5.2 Md and 7.8 Md plasmids




Concatemers of the 2.6 Md cryptic plasmid have been discovered in gonococci. 
A dimer was labelled a 5.2 Md plasmid and the trimer a 7.8 Md plasmid. Both 
can occur in beta-lactamase-producing and non-beta-lactamase-producing strains.

24.5 Md plasmid

This plasmid was reported first in 1975 (8) and subsequently has been found 
in both PPNG and non-PPNG but not in any other clinical species (21). The 
conjugative properties of the 24.5 Md plasmid were detected in PPNG. The 
G:C content of the 24.5 Md plasmid is 5096 with a low copy number of 2 3. 
The prevalence of this plasmid in gonococci is variable but is generally as low

• as 7 - 896. The plasmid does not carry any phenotypic characters apart from 
autotransferability and the sex factor. The plasmid mobilises transfer of other 
gonococcal plasmids such as the 3.2 Md and the 4.5 Md with high efficiency.

It can also be used to mobilise the penicillinase-producing plasmids to other 
bacterial species. As many as 1096 of cells carrying the 24.5 Md plasmid may 
act as conjugal donors. The 24.5 Md plasmid carries no detectable markers 
for drug, heavy metal or


13

ultraviolet resistance (18). Information about the restriction map of the 24.5

)ld plasmids is scanty but the structure of a limited number have been determined, 
and these experiments have revealed up to 30% variation in the 24.5 Md base 
sequences.

Resistance Plasmids

Strains of N. gonorrhoeae that contain plasmids coding for production 
of penicillinase were first isolated in U.K; and the U.S.A, and originated in 
West Africa and the Far East respectively. The plasmid from West Africa was 
a 3.2 Md plasmid, whereas the one from South-East Asia was a 4.4/4.5 Md plasmid 
there being differences in molecular size depending on conditions of preparation. 
The G:C ratio of both plasmids is 2:3 and is different from the G:C ratio of 
the chromosome of the gonococcus (24) indicating an exogenous origin. Molecular 
analysis and hybridisation studies showed that the 3.2 Md and 4.5 Md plasmids 
were identical except for a 2.1 kb fragment missing from the 3.2 Md plasmid.

The antibacterial action of penicillins and cephalosporins, (the beta-lactam 
antibiotics) is by preventing cross linking between acetylglucosamines and acetylmuramic 
acid during cell wall synthesis. Neisseria gonorrhoeae usually produce beta-lactamases 
that reduce or eliminate the toxic potential of this group of betalactam antibiotics 
by cleaving the amide bond of their lactam ring. Penicillinase is a special type 
of beta-lactamase which has substrate specificity for penicillins. The mode 
by which penicillinase acts on penicillin is shown in Fig. 1 (25).


RCONH

FIG 1. Inactivation of penicillin by beta-lactamase. The penicillin 
molecule (A) forms an acyl enzyme intermediate (B) with a serine 
residue in the active centre of the beta-lactamase. The hydrolyzed 
penicilloic acid derivative (C) is released (After 25).


DNA hybridization studies have shown that both gonococcal plasmids 
possess about 40co of transposable (TnA) sequence which includes the beta-lactamase 
gene or the bla gene. The beta-lactamase gene is commonly found on R plasmids 
of Enterobacteriaceae. Haemophilus influenzae and N. gonorrhoeae. Similarities 
have been observed between gonococcal beta-lactamase enzymes and those 
from H. influenzae and H. ducreyi.

Two additional beta-lactamase plasmids not associated with outbreaks 
of beta-lactamase N. gonorrhoeae infections were detected (24). Both plasmids 
are 4.6 kb in length. The restriction maps of both plasmids are similar except 
that the small Hind III - Bam HI fragment is 0.1 kb smaller than the equivalent 

fragment in the 4.5 i\Id plasmid (26).


16

Strains carrying the 4.5 Md plasmid are now found in all areas of the world 
including Europe, and West. Central and East Africa. The spread of the 3.2 Md 
plasmid has been limited to Europe and Central Africa.

The prevalence of PPNG is highest in Asia and sub-Saharan Africa but 
low in the developed world. In countries of the Asia-Pacific region, PPNG strains 
represent a large proportion of gonococcal isolates (5). In Indonesia, for example 
during 1980 to 1984 the prevalence of PPNG rose from 4 to 796 in STD clinic 
patients and from 17 to 3796 in prostitutes (27). In Thailand, 4876 of the gonococcal 
isolates showed decreased sensitivity to penicillin in 1971, and by 1983 the number 
of such isolates had doubled (28). In Bangkok, the prevalence of PPNG increased 
from 9% in 1978 to 4996 in 1981 (28). In Taiwan, 5496 of the N. gonorrhoeae 
strains isolated in 1985 were PPNG (29). In Korea, PPNG prevalence increased 
from 1% to around 3196 between 1981 and 1984 (30). In Japan, the prevalence 
of PPNG increased from 596 in 1980 to about 1596 in 1984 (31).

A similar dramatic spread in PPNG occurred in Africa. PPNG Strains 
that emerged in West Africa moved through to Central and East Africa. In 
Nigeria, PPNG strains were first detected in 1979 and by 1984, 8196 of the gonococci 
were PPNG (32). In Kenya (Nairobi), PPNG prevalence increased from 496 in 
1981 to 2296 in 1983 (33). In Tanzania, PPNG prevalence rose from 0.296 in 
1981 to 1996 in 1984 (34). In Ethiopia (Addis Ababa), the prevalence of PPNG 
rose from 5796 to 6196 between 1983 and 1984 (35). In Mozambique (Maputo), 
a prevalence study showed a 196 PPNG rate over a 5 week period (36). In South 
Africa (Durban), the prevalence of PPNG increased from 796 to 1296 between 
1977 and 1984 (37). In Zambia (Lusaka), PPNG prevalence rose from 296 to 

41% between 1982 and 1984 (38).


17

In contrast, there have been limited increases in PPNG in the developed 
countries. For example in the U.S.A., there was a slow increase in the number 
of PPNG cases up to 1980 followed by a sharp increase onwards (39). Even 
so the proportion of PPNG among total gonococcal isolates is still below 196. 
This low incidence probably indicates the effectiveness of early case detection 
and the use of alternative drugs in control programmes. In other countries 
such as the Netherlands and the U.K., although a number of cases have been 
reported, the overall incidence remains very low (5).

There is limited information about the prevalence and characteristics of PPNG 
in Zimbabwe and most of the information is limited to the Mbare Genitourinary 
Centre (GUC) in Harare. The Centre is a referral clinic, and therefore the 
data may not reflect the true situation for the country as a whole. In a small 
survey in 1982, 1098 of strains isolated from men in Harare were PPNG (40). 
Upto 1196 of the N. gonorrhoeae isolated at the Mbare Genitourinary Centre 
in 1983 were PPNG (41). An increase of such strains to 3796 was noted by the 
end of 1983 (42). Sixty per cent of N. gonorrhoeae strains isolated from women 
attending a genitourinary clinic in Harare were shown to be penicillinase producing 
143). These few statistics indicate that PPNG are present in Zimbabwe but 
the data are not representative of the whole of the country.

Other plasmids

A 2.9 Md plasmid has recently been associated with penicillin resistance in 
N.gonorrhoeae (26). It is slightly smaller than the 3.2 Md and has a transfer 
deficiency so it is found only in combination with the 24.5 Md plasmid which 
acts as a conjugative plasmid. A 25.2 Md plasmid has been shown to be associated 


18

with tetracycline resistance in _N. gonorrhoeae (44). The plasmid could have 
arisen as a result of a recombinational process between the 24.5 Md conjugative 
plasmid and a tet.M determinant. Recent studies have shown that the 24.5 Md 
conjugative plasmid and the 25.2 Md tetracycline resistant plasmid share >60% 
DNA sequence homology. The 25.2 Md plasmids have characteristics which 
differ from those of the 24.5 Md plasmid, but have retained the ability to move 
themselves and the 3.2 Md penicillinase-producing plasmids (44).

Interaction between factors responsible for penicillinase resistance.

The level of resistance to penicillin in microbial organisms is determined by 
the interplay of several factors. These include the rate of penicillin penetration 
of the outer membrane, the amount of penicillinase produced by the bacterium 
and the kinetic properties of the enzyme. Most studies have been done on E. coli 
but the results probably also apply to N. gonorrhoeae. The intrinsic resistance 
of plasmid-free cells is partially determined by the barrier to drug penetration 
posed by the outer membrane. Even in beta-lactamase producing cells, the 
expression of resistance is thus partly controlled by the surface layer. The 
drug itself also has an effect on its own penetration. In the case of beta-lactam 
drugs their ability to diffuse through the outer membrane is dependent on their 
charge and hydrophilic properties.

In addition, there is a direct relationship between the number of copies of the 
TEM beta-lactamase genes, the amount of penicillinase synthesised and the 
level of resistance (25). The rate of beta-lactamase production is thus dependent 


19

on those factors that influence plasmid copy number and therefore gene copies 
per cell. Thus there is an interaction of intrinsic resistance due to the presence 
of the outer membrane and the level of penicillinase which is genetically determined.

The affinity of an enzyme for a particular substrate also plays an important 
role in determining susceptibility of antibiotics to inhibitors. If an enzyme 
has a low Km value, then it has a high affinity for the substrate and vice versa.

Objectives of the study

The plasmid content of a bacterial strain is thought to be constant in 
a given geographic area and within a specified period of time (25). This fact 
makes the plasmid content of bacteria useful as epidemiological markers since 
bacterial strains causing a disease outbreak can be 'fingerprinted' using plasmid 
content. Since N. gonorrhoeae contain different sized plasmids, the plasmid 
profile was used to ascertain the most prevalent plasmid types which confer 
resistance in N. gonorrhoeae isolates from clinical cases presenting at chosen 
hospitals and centres in Zimbabwe. Thus the major objective of the project 
was to establish the plasmid types which confer resistance to penicillin in N. 
gonorrhoeae in Zimbabwe, and to determine the most commonly occurring plasmid 

associated with PPNG.


20

CHAPTER 2

MATERIALS AND METHODS

2.1 SPECIMEN COLLECTION




Patients with urethral discharge were identified by doctors and nurses 
upon arrival at the hospital or clinic. In Harare, most of the specimens were 
collected from patients who visited the Mbare Genitourinary Centre (GUC). 
Other specimens were collected from centres in the country covering seven 
provinces, as shown in Figure 2. No specimens were collected from Matebeleland 
South. A total of 865 specimens were collected. Of these specimens 837 were 
from males, 17 from females and 11 from babies.

In males, specimens were collected directly from the urethral discharge 
or from discharge induced by "milking" the urethra. If no discharge was obtained, 
an unmoistened thin sterile cotton swab was inserted into the distal urethra 
for approximately 2 cm and rotated gently.

In the case of females, the external urethral area was wiped dry with 
a sterile swab and discharge stimulated by applying pressure on the pelvis, 
the discharge was collected from the endocervix by using a swab and a ringing 
motion to help force the exudate from the endocervical glands. Eye swabs 
were taken from eyes of children who presented with ophthalmia neonatorum.

For the Mbare GUC and Bulawayo specimens, the swabs were streaked 
directly on plates of selective Thayer-Martin (TM media) (Appendix 1C) on 
site and were transported under conditions of increased CO2 and reduceo O2 
(candle jar) at ambient temperature.


KARIBA

MT. DARWIN 
¦> •

• KWE KWE

•GWERU

CHINHOYI0-? • HARARE 
#KADOMA r

eGUTG

MASVINGO

MUTAR

ZVISHAVANE,

3

I <

Fig.2 : Map showing areas where sample collection was done.


22

Swabs collected from other centres were transported in tubes of Amies 
or Stuarts transport media (Appendix ID and E) without environmental control.

ISOLATION OF N. gonorrhoeae

On arrival at the laboratory, swabs transported in Amies or Stuarts transport 
medium were removed and seeded onto TM plates. The swabs were rotated 
on the surfaces of the TM plates ensuring that all surfaces of the swabs came 
into contact v/ith media. Inoculated plates were placed as soon as possible 
man environment of 70% humidity'arid 5 -'1096 CO2. Stroh’an environment 
was created by using a candle jar, CO2 generating kit or a CO2 incubator. 
Incubations were at 37°C and plates were examined after 24 hours for colonies 
typical of N. gonorrhoeae. Typical colonies are about 1 mm in diameter, and 
may vary from transparent to opaque, and may be raised convex to flat.

2.3 IDENTIFICATION TESTS




Typical colonies were examined by the Gram stain, oxidase test and carbohydrate 
fermentation tests.

I J y

2.3.1 Gram stain




A loopful of the bacterial culture was placed on a clean slide and spread into 
a thin film. The slide was fixed by passing through a flame. It was flooded 
with methyl violet for 30 seconds, then iodine for 10 seconds, washed with 9596 
alcohol and then counterstained with carbolfuschin for 5 seconds. The stained 
preparations were then examined under oil-immersion objective. Groups of 
Cram-negative, kidney shaped diplococci with flattened margins were indicative 
of Neisseria spp. (45).


23

2.3.2 Oxidase test




A strip impregnated with the oxidase reagent (p-aminodimethvlaniline-

oxalate 1%) was smeared with a bacterial culture. A positive test was indicated

by a purple colouration within 5 seconds. The oxidase test assesses the ability

of a microbe to oxidise certain aromatic compounds such as phenol to form 
coloured end products (46).

2.3.3 Acid production from carbohydrates




The carbohydrates used for the test were glucose, maltose and sucrose.

Gonococcal Agar base (Appendix 1A) containing phenol red as the indicator 
was used as the basal medium. The medium was sterilised by autoclaving at 
121°C for 15 minutes and after cooling to 45°C, an appropriate amount of filter 
sterilised carbohydrate was added to give a final concentration of 1%. The 
medium was then poured into Petri dishes. The plates were inoculated with 
a fairly heavy inoculum of the isolates and incubated in 5 - 10% CO2 at 37°C 
lor 24 hours. A yellow colour indicated utilisation of the sugar with acid production 
(47).

2.3.4 Antibiotic sensitivity tests




Plates of chocolate agar were lawned with a colony of each of the isolates.

Antibiotic discs incorporated with tetracycline (25 ug), augmentin (30 ug), erythromycin 
(5 ug) and penicillin G (2U) were placed on each plate. The plates were then 
incubated at 37°C for 24 hours and examined for zones if inhibition of growth 

around each disc.


24

2.3.5 Beta-lactamase activity




Beta-lactamase was detected using the bromocresol purple reaction, using 
a Commercial stip (Mast Laboratories, Merseyside, U.K.). A 48 hour culture 
of N. gonorrhoeae was collected using a curved end of a capillary tube ensuring 
that several colonies were touched and placed on a strip moistened with 0.9% 
saline. This w as done in order to avoid misleading results due to occasional 
beta-lactamase producing colonies. The cells were spread onto the test area 
of the beta-lactamase strip. A change in colour from purple to yellow within 
10 minutes indicated beta-lactamase activity (36, 37).

2.4 REFERENCE STRAINS:




Neisseria gonorrhoeae reference strains were provided by Inga Lind of

the WHO Collaborating Centre for Research in Gonococci, Copenhagen, Denmark. 
The strains were SSI-PI (2.6 + 3.2 Md), SSI-P2 (2.6 + 4.5 + 24.5 Md) and SSI-P3 
(7.8 Md). To regrow the strains, 2 ml of nutrient broth (Appendix IF) were 
added to the lyophilised cultures. The suspensions were inoculated on Thayer- 
Jlartin (Appendix 1C) and chocolate agar (Appendix IB) and incubated in 5 - 10% 
CO2 at 37°C for 24 hours.

2.5 ISOLATION OF PLASMID DNA




Plasmid DNA was isolated using selective alkaline denaturation (NaOH- 
SDS) (Appendix 2b) of high molecular weight DNA (40, 41, 42, 43, 50, 51, 52, 
53). In this procedure covalently closed circular plasmid DNA remains double 
stranded and suspended in the supernatant while chromosomal DNA denatures 
to form an insoluble clot (38, 54).


25

Isolates and reference strains were grown in pure culture on chocolate

agar (Appendix IB) (penicillin-sensitive) or chocolate agar (Appendix 1C) containing 
Img/1 ampicillin (penicillin resistant). The purpose of plating the cultures onto 
chocolate agar that contained ampicillin was to enhance the selection of penicillin 
resistant plasmids. Growth from the plates were scrapped with an L shaped 
glass rod into 1.5 ml of phosphate buffered saline. (PBS, 50 mM, pH 7.2) in Eppendorf 
tubes. This was centrifuged at 10 000 revolutions per minute (rpm) for 15 seconds 
at room temperature in a microfuge centrifuge. The supernatants were tipped 
off and the bacterial pellets resuspended each in 100 pl of freshly prepared 
solution containing lysozyme (1 mg/ml) in 50 mM Tris-HCl; pH 8.0 (Appendix 
2a). The reactant solutions were placed on ice for 30 minutes. Two hundred 
microlitres of 0.2 M NaOH - 1% SDS (Appendix 2b) were added, and the contents 
of the tube mixed by gentle vortex before being incubated on ice for a further 
5 minutes. One hundred and fifty microlitres of 3 M sodium acetate (Appendix 
2c) was then added to each Eppendorf tube and incubated at 20°C for 60 minutes. 
The contents of the Eppendorf tubes were centrifuged at 10 000 rpm for 5 minutes. 
Forty microlitres of each supernatant was transferred to a clean Eppendorf 
tube and 1 ml of cold absolute ethanol added. The mixtures were left at -20°C 
for 30 minutes and then centrifuged at 10 000 rpm for 5 minutes. Each pellet 
was dissolved in 100 pl of 100 mM sodium acetate, 5 mM Tris-HCl (Appendix 
2d); pH 8.0 to which 200 pl of cold absolute ethanol was added. The tubes were 
placed at -20°C for 30 minutes and centrifuged at 10 000 rpm for 2 minutes.

The supernatant was removed and tubes drained well to remove excess ethanol 
while taking care not to dislodge the pellet. The pellets were each dissolved 
in40 pl of 30 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 8.0 (39) (Appendix 
2e). Two microlitres of RNase (1 mg/ml) (Appendix 2f) were added to each 
Eppendorf tube. The tubes were incubated at 37°C for 30 minutes.


26

To check for purity, 10 pl of the preparation was diluted to 100 pl in TE buffer 
and scanned spectrophotometricallv in the range 220 - 300 nm. The rest of 
the extract was stored at -20°C until required (55).

2.6 PREPARATION OF GELS




Agarose gels were prepared by dissolving 0.8 g of agarose type II (Sigma 
Chemical, U.S.A) in 100 ml of Tris - acetate - EDTA buffer (IX) (Appendix 
2i) in a conical flask. This was heated to boiling until no solid pieces of agarose 
were discerned, and then cooled to 55 - 60°C. The open ends of the gel trays 
were sealed with masking tape. A slot former was then placed into position. 
The trays were placed on a level surface, the agarose gel poured and allowed 
to solidify for at least 15 minutes. The slot formers were slowly pulled out 
starting with one end. The masking tape was removed and the gel plates carefully 
placed in the electrophoresis tanks. Tris-acetate-EDTA buffer (Appendix 2i) 
was added to the tanks until the gels were submerged.

2.7 ELECTROPHORESIS




Five microlitres of gel loading buffer (50X) (Appendix 2g) was added to 
15 pl of the DNA sample. The DNA and dye were mixed by vortex. The gels 
were then loaded with samples (20 pl) and electrophoresis was carried out at 
80 volts, 50 mA and 50 Watts for 2.5 hours. Molecular weight markers were 
included in each run.


27

The gels were stained with ethidium bromide (10 mg/ml) (.Appendix 2h)

for 10 minutes. They were then washed with distilled water and observed under

aUV transilluminator for demonstration of DNA bands. Photographs of the

gels were taken using a polaroid camera.


28

CHAPTER 3

RESULTS

3.1 ISOLATION OF N. gonorrhoeae

The number of specimens collected and the isolation rates are shown in 
Table 1. N. gonorrhoeae was isolated from 436 (50%) of the total specimens 
collected. Table 1 further shows that Mashonaland East province recorded 
the highest isolation rate (75%) and Manicaland the lowest (31%). Masvingo 
province recorded a higher isolation rate than the Midlands, Mashonaland (Vest 
and .Manicaland provinces. However, in interpreting the results, one should 
take into account that the percentages may not represent the true prevalences 
since the number of specimens sampled from each province were different.

Table 1. Overall isolation rates for the different provinces for the period

July 1987 - July 1989.

Total No. of

N. gonorrhoeae

No. (%) R Plasmid

Province

specimens

collected

isolates; No. (%)

bearing isolates



Manicaland

144

45 (31)

5

(11)

Masvingo

19

10 (53)

3

(30)

Midlands

71

26 (37)

3

(12)

Mashonaland West

213

90 (42)

27

(30)

Mashonaland East

220

166 (75)

92

(55)

Mashonaland Central

26

1 (4)

1

(100)

Matebeleland North

172

98 (57)

16

(16)

TOTAL NO.

865

436 (50)

147

(34)




29

3.2 ANTIBIOTIC SENSITIVITY TESTS




Table 2 shows the results of sensitivity tests using penicillin G, tetracycline, 
erthromycin and augmentin. Most isolates were resistant to Penicillin G but 
were sensitive to the other antibiotics. Only two isolates were resistant to 
augmentin while four and six isolates were resistant to erthromycin and tetracycline 
respectively.

The highest number of isolates which were resistant to Penicillin G were from 
Mashonaland East followed by those from Mashonaland West. Fewer isolates 
from Manicaland, Midlands and iMatebeieland North were resistant to Penicillin G.

Some isolates displayed resistance to more than one antibiotic. For example, 
one isolate each from Manicaland, Mashonaland West and Mashonaland East 
were resistant to both penicillin G and erythromycin. An isolate each from 
Mashonaland West, Matebeleland North and Mashonaland East were resistant 
to both penicillin G and tetracycline. One isolate from Mashonaland West was 
resistant to both penicillin G and augmentin.

Table 2. Results obtained for antibiotic resistances for the regimens used

in the treatment of gonorrhoea.

No. (%) resistance to

Province

penicillin G

augmentin

tetracycline

erythromycin

Manicaland

5 (11)

0

(0)

1

(2)

1

(2)

Masvingo

3 (30)

0

(0)

0

(0)

0

(0)

Midlands

3 (12)

0

(0)

1

(4)

0

(0)

Mashonaland West

27 (30)

1

(1)

2

(2)

1

(1)

Mashonaland East

99 (60)

1

(1)

1

(1)

2

(1)

Mashonaland Central

1 (100)

0

(0)

0

(0)

0

(0)

Matebeleland North

16 (16)

0

(0)

1

(1)

0

(0)




30

3.3 COMPARISON OE BETA-LACTA.M ASE ACTIVITY AND PENICILLIN G 
RESISTANCE




The prevalence of beta-lactamase activity in isolates is presented in Table 3 
which also compares the results for beta-lactamase activity with those for 
resistance to penicillin G. The largest percentage of strains showing beta-lactamase 
activity were from Mashonaland East and the lowest from Manicaland. Seven 
beta-lactamase negative strains from Mashonaland East were resistant to penicillin G. 
All beta-lactamase producing strains isolated from the other provinces were 
also resistant to penicillin G.

Table 3. Comparison of numbers of beta-lactamase positive strains and those 
that were resistant to penicillin G.

Province

No. (96) beta-lactamase

No. (96) resistance

positive

to penicillin G



Manicaland

5

(11)

5

(11)

Masvingo

3

(30)

3

(30)

Midlands

3

(12)

3

(12)

Mashonaland West

27

(30)

27

(30)

Mashonaland East

92

(55)

99 (60)

Mashonaland Central

1 (100)

1 (100)

Matebeleland North

16

(16)

16

(16)




31

3.4 PLASMID DNA ANALYSIS




The UNA preparation was pure with an A260:A280 ratio of 1.48.

The 2.6 Md cryptic plasmid was the only plasmid detected in the 289 non-PPNG

isolates analysed.

The plasmid profiles of the 147 PPNG isolates are presented in Table

4. The results indicated that 144 (98%) harboured the 2.6 Md cryptic plasmid;

56 (3896) the 3.2 Md plasmid, 66 (45%) the,4.5 Md plasmid and 61 (41%) contained

I 
the 24.5 Md transfer plasmid. The 7.8 Md trimer plasmid existed in combination

with the 2.6 Md cryptic plasmid in 58 (39%) cases and in 17 (12%) cases without

the cryptic plasmid but with other plasmids. The ratio of 3.2 Md to 4.5 Md

plasmid was 5:6.

¦

Table 4. Plasmids detected in PPNG strains by agarose gel electrophoresis..

Province

% PPNG

2.6

Plasmid sizes (Md)

Number (%)

24.5

3.2

4.5

Manicaland

5

(11)

4

(80)

1 (20)

3 (60)

2 (40)

Masvingo

3

(30)

3

(100)

2 (67)

1 (33)

3 (100)

Midlands

3

(12)

3

(100)

1 (33)

2 (67)

2 (67)

Mashonaland (vest

27

(30)

25

(93)

8 (30)

12 (44)

1

6 (22)

Mashonaland East

92

(55)

92

(100)

37 (40)

38 (41)

38 (41)

Mashonaland Central

1

(100)

1 (100)

1 (100)

0 (0)

0 (0)

Matebeleland North

16

(16)

16

(100)

6 (38)

10 (63)

10 (63)

TOTAL

147

(34)

144

(98)

56 (38)

66 (45)

61 (41)




32

3.5 VARIOUS PLASMID COMBINATIONS




A summary of the various plasmid combinations is presented in Table 5.

As the 2.6 Md plasmid is cryptic and was present in all combinations, attention 
will only be focussed on R-bearing and conjugative plasmids. The most prevalent 
plasmid combination was the 4.5 Md with no conjugative plasmid (36 isolates), 
followed equally by the 3.2 .Md plasmid with no conjugative plasmid and the 
¦1.5 Md plasmid + 24.5 conjugative plasmid (both 29 isolates). Eighteen isolates 
did not carry either the R-bearing plasmids or the conjugative plasmid while 
13 isolates only carried the conjugative plasmid. There was an isolate with 
a rare combination of the 3.2 Md + 4.5 Md + 24.5 Md plasmids and 7 isolates 
with a combination of 3.2 Md + 4.5 Md + Co + 24.5 Md plasmids.

Table 5. Plasmid combinations obtained for all the provinces.

Number of isolates bearing the plasmid

combination size (Md)

Province

0

3.2

3.2 + 24.5

4.5

4.5 + 24.5

24.5

3.2 + 4.5 
+ 24.5 + (Co)

Manicaland

0

1

0

2

1

1

0 (0)

Masvingo

0

0

2

0

1

0

0 (0)

Midlands

0

0

1

1

1

0

0 (0)

Mash. West

5

5

2

9

2

1

1 (3)

Mash. East

9

27

10

18

20

8

0 (3)

Mash. Central

0

1

0

0

0

0

0 (0)

Mat. North

4

0

2

6

4

4

0 (1)




Examples of the various plasmid combinations are shown in Figures 3 - 6. For 
example. Figure 3 lane 9 shows the 2.6 Md + 3.2 Md + 4.5 Md + 24.5 Md combination.

The same combination plus the 7.8 Md plasmid is shown in Figure 6 lanes 3 and

5. The 2.6 Md + 4.5 Md + 7.8 Md + 24.5 Md combination is demonstrated in

Figure 3.land 6, Figure 4 lane 8 and Figure 5 lane 8. Figure 6 lane 6 demonstrates

the 3.2 Md + 7.8 Md combination whilst Figure 5 lane 12 shows the 4.5 Md +

24.5 Md combination and the same Figure (Figure 5) lane 2 demonstrates the

2.6 Md + 4.5 Md combination.


34

Molecular weight (Md)

Figure 3: Electrophoretic pattern of plasmid extracts from

N. gonorrhoeae

Lane 1 = Reference Strain; 2.6 + Co + 24.5 Md

Lane 2 = isolate from Mash. West; 2.6 + Co + 24.5 Md plasmids

Lane 3 = isolate from Masvingo Province; 2.6 Md plasmid only

Lane 4 = isolate from Mash. East; 2.6 + 4.5 + 24.5 Md plasmid

Lane 5 = Reference Strain; 2.6 + 4.5 + Co + cm + 24.5

Lane 6 = isolate from Mash. East; 2.6 + 4.5 + Co + 24.5 Md plasmid

Lane 7 = isolate from Masvingo Province; 2.6 + 4.5 + Co + 24.5 Md plasmid

Lane 8 = isolate from Mashonaland East; 4.5 Md plasmid only

Lane 9 = isolate from Mash. West; 2.6 + 3.2 + 4.5 + Co + 24.5 Md

Co = trimer of cryptic plasmid

cm

chromosomal


Figure 4: Electrophoretic pattern of plasmid extracts from

N. gonorrhoeae

Molecular weight (Md)

Lane 1 = Reference Strain; 2.6 + 4.5 + Co + 24.5 Md

Lane 2 = isolate from Mash. East. 2.6 + Co + 24.5

Lane 3 = isolate from Mash. West; 2.6 + 3.2 + Co + 24.5

Lane 4 = isolate from Mash East; 2.6 + Co + 24.5

Lane 5 = isolate from Mash. East; 2.6 + Co + 24.5 Md only

Lane 6 = isolate from Manicaland; 2.6 + 3.2 + 4.5 + Co + 24.5 Md only

Lane 7 = isolate from Mash. East; 4.5 + Co

Lane 8 = isolate from Mash. East; 2.6 + 4.5 + Co + 24.5

Lane 9 = isolate from Mash. West; 2.6 + 3.2 + 4.5 + Co + 24.5

The contaminating RNA was removed by an RNase treatment.

Co = trimer of cryptic plasmid.


36

Molecular weight (Md)

Figure 5: Electrophoretic pattern of plasmid extracts from

N. gonorrhoeae

Lane 1 = Reference Strain; 2.6 + 4.5 + Co + 24.5 Md only

Lane 2 = isolate from Mash. East; 2.6 + 4.5 Md only

Lane 3 = isolate from Mash. East; 2.6 + 4.5 + Co

Lane 4 = isolate from Mash. East; Co

Lane 5 = isolate from Mash. East; 2.6 + 4.5 + Co + 24.5 Md

Lane 6 = isolate from Mash. East; no plasmids

Lane 7 = isolate from Mash. East; no plasmids

Lane 8 = isolate from Mash. East; Co only

Lane 9 = isolate from Mash. East; no plasmids

Lane 10 = isolate from Mash. East; 2.6 + 4.5 + Co + 24.5

Lane 11 = isolate from Mash. West; 2.6 + 4.5 + Co + 24.5

Lane 12 = isolate from Mash. East; 4.5 Md + 24.5 Md

Lane 13 = isolate from Mash. East; 2.6 + 4.5 + Co

Lane 14 = Reference strain ; 4.5 + 24.5 Md plasmids

Co

trimer of the cryptic plasmid.


Figure 6: Electrophoretic pattern of plasmid extracts from

Molecular weight (Md)

N. gonorrhoeae

Lane 1 = tracking dye

Lane 2 = isolate from Mash. East; 2.6 + 3.2 + (Co) + 24.5

Lane 3 = isolate from Mash. East; 2.6 + 3.2 + 4.5 + Co + 24.5

Lane 4 = isolate from Mash. East; 2.6 + 3.2 + 4.5 + Co

Lane 5 = isolate from Mash. West; 2.6 + 3.2 + 4.5 + Co + 24.5

Lane 6 = isolate from Mash. East; 3.2 + Co

Lane 7 = isolate from Mash. East; 2.6 + 3.2 + Co

Lane 8 = isolate from Manicaland; 2.6 + 3.2 + 4.5 + Co

Co

trimer/dimer of cryptic plasmid


38

CHAPTER 4

DISCUSSION

An attempt was made to cover as much of Zimbabwe as possible. However, 
because only a few representative centres could be sampled, there was emphasis 
on larger centres of population, rather than rural areas where 7096 of the population 
lives.

There were also differences in the way the samples were handled before 
culture. Some samples such as from Harare were directly cultured on collection. 
Incases where centres were too remote, the investigator physically visited 
the hospital and cultured the samples. For those centres where transportation 
by road was possible, samples were cultured in transport medium. However, 
in most cases transportation of this medium was unreliable and many samples 
reached the laboratory in an unsatisfactory state.

The isolation rates for Mashonaland East and Matebeleland North were 
higher than the other provinces. This might be attributed to the fact that the 
collection centres in these provinces were in close proximity to the laboratories. 
For instance, in Mashonaland East Province, Mbare GUC is close to the main 
laboratories at Parirenyatwa Hospital. Furthermore, Mbare GUC is a referral 
Clinic for STD and so a higher isolation rate might be expected. The same 
reasons apply for the high isolation rate for Matebeleland North where specimens 
were collected from Khami Clinic which is very close to Mpilo Hospital.


Although more samples were obtained from .Midlands and Manicaland 
provinces than .Masvingo province: the Masvingo specimens were cultured on 
site while those from the other two provinces were transported in refrigerated 
trucksand reached the laboratory at the University Medical School in a nonviable 
state. This is reflected in the higher isolation rate obtained for the Masvingo 
specimens. Whether PPNG or particular plasmid-bearing strains are more susceptible 
to the adverse conditions associated with the sampling methods used in some 
areas is not known. Thus the conclusions drawn from the findings must be viewed 
with this in mind.

More isolates were resistant to penicillin G than to the other antibiotics 
tested. The high penicillin resistance percentage for Mashonaland East was 
to be expected since most patients who visit the Mbare GUC are referral patients, 
it is likely that penicillin would have been used as first treatment at peripheral 
centres. However, even for the other provinces penicillin resistance was relatively 
high ranging from 1196 for Manicaland to 3 0 96 for Masvingo and Mashonaland

l.est. For those isolates from Masvingo although a small population of N. gonorrhoeae 
was screened, every 1 in 5 of the isolates was resistant to penicillin G. Seven 
isolates from Mashonaland East were beta-lactamase negative but resistant 
to penicillin G. This is indicative of resistance to penicillin G through chromosomal 
mutations. Similar results are documented for the work done by Ison (13) and 
Sparling (12).




For augmentin, this is a surprising result since augmentin resistance is 
known to be correlated with chromosomal resistant gonorrhoeae. It would have 
been expected that all the seven beta-lactamase negative penicillin resistant 
isolates would also be augmentin resistant. Evidence from (MIC) studies (56) 
also indicate that resistance to augmentin as well as tetracycline and erythromycin 
could be expected in local gonococci.


4U

This study showed a PPNG rate of 34%. whilst similar studies by Marowa 
(57) and Mason (58) indicated PPNG rates of 40% and 67% respectively. The 
figure is comparable to those of other African countries. For example. 22% 
for Kenya, (33) 25% for Zambia (38). 21% for Gabon (59), 49% for Ethiopia (35), 
51% for Rwanda (60), 23% for Libya (5). The figure is much higher than 12% 
for South Africa (Johannesburg) (37) and much lower than 81% for Ghana (5). 
Given that these figures are probably from two years ago, the present figures 
are most likely to be higher than the ones given.

Resistance to tetracycline can be plasmid-mediated and associated with 
the 25.2 Md conjugative plasmid, or chromosomally mediated. However,- in 
this study the 25.2 i\Id plasmid was not observed from any of the isolates even 
the one that had resistance to tetracycline. A possible source of the tetracycline 
resistance plasmid could be commensal Neisseria spp. that were documented 
by Prere (61) to be sources of resistance genes transferrable to pathogenic species.

The plasmid DNA preparation was relatively pure as shown by the A260:A280 
ratio of 1.48. For good DNA preparations, the A260:A280 ratio is normally 
greater than 1.7 (52). Plasmid analysis of PPNG strains indicated that the 2.6 
Mdexisted in 98% of the isolates. This is in agreement with earlier work (23) 
and that by Johnson (63) in the Gambia which demonstrated that the 2.6 Md 
plasmid existed in both PPNG and non-PPNG. This is also in agreement with 
work done by Lefevre (62) in France and Mason and Gwanzura (58) in Harare, 
Zimbabwe who indicated the presence of the 2.6 Md plasmid in all the PPNG 

tested.


41

Contrary to this study's findings Monayar 8 et al(64) indicated that only 
83% of N. gonorrhoeae isolated in their study harboured the 2.6 ?Id plasmid. 
Moreno et al (39) in his studies in Chile also indicated that only 67% of the 
PPNG strains tested carried the 2.6 Md plasmid. Cannon (8) showed that some 
autotypes of gonococci do not necessarily have to harbour the cryptic plasmid.

Plasmids of major interest in this study were the 3.2 Md and the 4.5 Md 
plasmids. The two plasmids were found to equally confer penicillin resistance 
to the N. gonorrhoeae strains isolated. This is contrary to an earlier study 
158) which showed the 4.5 Md plasmid as the predominant plasmid responsible 
for penicillinase production. The 4.5 Md plasmid in the PPNG strains shows 
that this plasmid is now spread to almost all parts of the world and is no longer 
confined to the Far East (23). It has been shown for example to be the predominant 
plasmid in the majority of PPNG isolates from Kenya (66) and Zambia (65). 
The 3.2 Md plasmid has been shown to be the predominant plasmid in PPNG 
isolates from West Africa (Frost et al, 59; Franceville and Osoba, 32; Bogaerts, 
60). Results from this study indicate that the plasmid might be spreading to 
Southern Africa.

The 24.5 Md conjugal plasmid was observed in 41% of the strains. It was 
observed in most cases in combination with the 2.6 Md plasmid, 3.2 Md or 4.5 
Md plasmids but in a few cases on its own. The figure is high and can probably 
explain the rapid spread of R-plasmids through the local gonococcal population.


42

Previous work (23) has shown that the 24.5 Md conjugative plasmid does not 
exist in all strains of PPNG and that its frequency is as low as 7 - 8°o.

Reference is made to Table 5 which shows the overall plasmid combinations 
obtained in the study. The most frequent combination was the 2.6 Md + 4.5 Md; 
then the 2.6 Md + 3.2 Md and the 2.6 Md + 3.2 Md + 24.5 Md. Similar studies 
in both Gabon and Rwanda (60) showed that the 4.5 Md + 24.5 Md and the 3.2 
Md + 24.5 Md were the most common plasmid combinations. A study on isolates 
from other African countries was carried out by Odugbemi (66). Results of 
the study demonstrated that the 2.6 Md + 3.2 Md combination existed in isolates 
from the Central African Republic, Zambia, Zaire and the Ivory Coast.

Eight isolates in this study harboured both the 3.2 Md and 4.5 Md plasmids. 
There is one similar case reported from Rwanda. An isolate should not be able 
to carry the two plasmids as they are incompatible. The results should therefore 
have been checked for authenticity by cloning the isolates in order to demonstrate 
that the results might not be due to double infection.

Conclusions

The study has demonstrated that the plasmid types that confer resistance to 
penicillin in N_. gonorrhoeae in Zimbabwe are the 3.2 Md and 4.5 Md plasmids. 
The study has also shown that the 4.5 Md plasmid and 3.2 Md plasmids existed 
in a ratio of 6:5. Although samples were collected from as many places as possible 
the data may not represent the National Status of the prevalence of PPNG

in Zimbabwe.


43

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and characterisation of plasmid DNA. Journal of Bacteriology 
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plasmid isolation. A laboratory experiment for intermediate and 
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57. Marowa, E. 1987 Latif, A. S., Paraiwa, E., Gutu, S. and Machachi, N.




The treatment of gonorrhoea in male and female patients with augmentin, 
penicillin and probenecid. Abstract, 5th African Regional Conference 
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serogroups, and auxotvpes of Neisseria gonorrhoeae from Harare, 
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Auxotvpes and serogroups of penicillinase-producing and non-penicillinase 
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Venereal Diseases 59 41 - 43.


1

53

APPENDICES

APPENDIX 1 :

Preparation of media

A. Gonococcal (GC) Agar Base




Gonococcal Agar Base comprised of:

Formula

g£l

pH approximately 7.2

Selected peptone mixture

10.0

Bacteriological peptone

5.0

Sodium chloride

5.0

Soluble starch

1.0

Potassium hydrogen orthophosphate

1.0

Di-potassium hydrogen orthophophate

4.0

Agar A (RM 10)

10.0



B. Chocolate agar




Thirty eight grams of GC agar base (Appendix 1A) were suspended in 1 litre 
of distilled water and mixed thoroughly. The medium was autoclaved at 121 °C 
for 15 minutes, and cooled to 50°C. Sterile sheep blood, 5 - 7% was added to 
the agar base and mixed thoroughly. The blood agar was held at 80°C with 
occasional mixing until it developed a chocolate brown colour and then poured 
into plates.


54

C. Thayer-Martin (TM)




Thirty eight grams of GC agar base were suspended in 1 litre of distilled 
water and autoclaved at 121 °C for 15 minutes. The GC agar base was cooled 
to50°C. Sterile sheep blood. 5 - 796 was added and mixed thoroughly. This 
was held at 55°C and 4 GC growth supplement tablets (Mast Laboratories, Merseyside, 
U.K.) and 10 ml of 2°6 sterile glucose were added aseptically. After the supplement 
was dissolved in suspension. 4 MS5 VCT tablets were added and the bottle inverted 
and mixed well to achieve complete dispersal before pouring the plates.

D. Amies Transport Media




Ingredients ff/1

Charcoal 10-0

Sodium chloride 3.0

Potassium chloride 0.2

Disodium hydrogen phosphate 1.15

Potassium dihydrogen phosphate 0.2

Sodium thioglycollate 1-0

Calcium chloride (anhydrous) 1.0

Magnesium chloride 0.1

AgarA(RMlO) 5.0

pH approximately 72.

The above formula was provided in sachets. The contents of each sachet were 
suspended in 200 ml of distilled water and boiled to dissolve the agar completely.


The boiled agar was dispensed into bijoux bottles and then autoclaved at 121 °C 
for 15 minutes. The bijoux bottles were inverted to ensure even distribution 
of the charcoal.

E. Stuarts Transport Media




Ingredients g/1

sodium glycerophosphate 10.0

sodium thioglycollate 1.0

calcium chloride 0.002

Agar 2.0

pH 7.4

Sixteen grams of the formula were suspended in 1 litre of distilled water and 
boiled to dissolve completely. This boiled agar was dispensed into bijou bottles 
and then autoclaved at 121 °C for 15 minutes. The bottles were inverted to 

ensure complete mixing.

F. Nutrient Broth




Ingredients

Bacto beef extract

Bacto peptone

g/1

3

pH approximately 6.8

Eight grams of nutrient broth were suspended in 1 litre of distilled water, and 
dispensed in 20 ml volumes into MaCartney bottles and autoclaved at 121 °C 

for 15 minutes.


5(5

APPENDIX 2.

Preparation of Buffers

a. Lvsis Buffer

Ingredients

g/100 ml

200 miH Glucose

3.6

40 mN EDTA

1.36

100 mM Tris-IICl, pH 8.0

1.21



Distilled water

Stock solutions of the lysis buffer were prepared and stored at 4°C. The lysis 
solution was prepared fresh by mixing 1 ml of lysis buffer and 1 mg of lysozyme.

b. NaOH - SDS

Ingredients

0.4 M NaOH

296 SDS

g/100 ml

1.6

2.0

The two solutions were prepared separately and mixed in equal amounts just

before use. (Final concentration 0.2 M NaOH: 196 SDS).

c 3 M sodium acetate

3 M sodium acetate, pH 4.8 (24.61 g/100 ml)

Anhydrous sodium acetate was used and the pH achieved using glacial

acetic acid.


57

0.1 \1 sodium acetate (anhydrous)

0.8 g/100 ml

0.5 M Tris-lICl. ph 8.0

0.6 g/100 ml



TES

Ingredients

g/100 ml

0.03 M Tris-HCl

0.36

0.05 M NAC1

0.29

0.005 M EDTA

0.17



pll was adjusted to 8.0 by adding IM NaOH.

RNase

Five milligrams of RNase A was dissolved in 5 ml of distilled water and 
boiled in a waterbath of 10 minutes prior to storage. This destroys DNase 
activity. The RNase was dispensed into 0.1 ml aliquots and stored at 
-20°C.

Gel loading buffer (50X)

50°o sucrose

0.5 g/ml

0.2% Bromophenol blue

0.002 g/ml

The buffer was stored at -20°C.

Ethidium Bromide (10 mg/ml) •

One hundred milligrams of ethidium bromide was dissolved in 10 ml of 

distilled water and stored at 4°C.


58

Stock running buffer

Tris-acetate - EDTA buffer

Ingredients

g/l

Tris base

48.4

sodium acetate

16.4

EDTA

7.4



The ingredients were dissolved in 1 litre of distilled water and pH adjusted 
to 7.8 by addition of 0.1 M HC1. For use this was diluted 1 in 10. The 
buffer was stored at room termperature.