SUSCEPTIBILITY OF SALMONELLA TYPHI ISOLATED FROM STOOL SAMPLES OF SYMPTOMATIC PATIENTS TO ROVTINE ANTIBIOTICS ATTENDING YUSUF DANTSOHO MEMORIAL HOSPITAL, KADUNA.
SUSCEPTIBILITY
OF SALMONELLA TYPHI ISOLATED FROM STOOL SAMPLES OF SYMPTOMATIC PATIENTS TO
ROVTINE ANTIBIOTICS ATTENDING YUSUF DANTSOHO MEMORIAL HOSPITAL, KADUNA.
ABSTRACT
Salmonella species are gram negative facultative
anaerobic rods. The study was carried out to determine the susceptibility
pattern of Salmonella typhi to
selected routine antibiotics. A total of 100 stool samples were collected from
Yusuf Dantsoho Memorial Hospital, Kaduna. Out of the total of 100 stool samples
cultured, 13 (130) Salmonella
isolated were identified by their colony morphology and biochemical tests of
which 7 (53.84%) were Salmonella typhi
and 6 (46.16%) were Salmonella
typhimurium. Out of the 13 Salmonella
isolates 5 (38.46%) were from males and 8 (61.54%) were from females. All the
isolates of Salmonella typhi were
subjected to antibiotic susceptibility testing using 10 antibiotics commonly
used for the treatment of typhoid fever Augmentin (25mg), Gentamycin (10mg), Pefloxacin (10mg), Tarivid (30mg), Streptomycin (30mg), Septnin (30mg), Chloramphenicol (30mg), Sparfloxacin (10mg), Ciprofloxacin (10mg) and Amoxacillin (30mg). the Isolates showed
resistance to 4 out of 10 Antibitics used (Chloramphenicol, Septrin, Augmentin
and Amoxicillin) but were sensitive to Ciprofloxacin, Pefloxacin, Gentamycin,
Tarivid, Streptomycin and Sparfloxacin with 21mm, 20mm, 16mm, 17mm, 21mm and
19mm as their mean zones of inhibition respectively. All isolates of Salmonella typhimurium were resistance
to 4 out of 10 antibiotics which are Chloramphenicol, Septrin, Augmentin and
Amoxicillin but sensitive to Ciprofloxacin, Pefloxacin, Gentamycin, Tarivid,
Streptomycin and Sparfloxacin with 19mm, 22mm, 17mm, 18mm, 10mm and 16mm
respectively. The results of this study suggest that only the Fluoroquinolones
are effective against the typhoid bacteria and the results call for nationwide
surveillance programme to monitor microbial trends and antibiotic resistance pattern
in Nigeria.
CHAPTER
ONE
INTRODUCTION
Salmonella
species member of the family Enterobacteriaceae are gram negative facultative anaerobic
rods. The genus Salmonella contain two
species S. enterica and S. bongori.S.enterica contains six subspecies which are S. enterica subspecies enterica,
S. enterica subspecies diarizonae, S.
enterica subspecies arizonae and S. enterica subspecies indica. Most of the isolates that cause
disease in human and her mammals belong to S.
enterica subspecies enterica(Center
for food security and public health, 2011).
Salmonella
enterica serotype typhi (S.typhi) inhabits the lymphatic tissues if the small intestine,
liver, spleen and blood stream of infected humans, S. typhi is the causative
agent of typhoid fever (David, 2013).
Salmonella typhi
has combination of characteristics that makes it an effective pathogen. This
species contains an endotoxin typical gram – negative organisms, as well as the
Vi antigen which is thought to increase virulence. It also produces and excretes
a protein known as “Invasin” that allows non-phagocytic cells to take up the
bacterium, where it is able to live intracellularly. It is also able to inhabit the oxidative burst of leukocytes
making innate immune response ineffective (David, 2003).
Ineffective
with Salmonella bacteria cause
typhoid fever in people if they are infected with Salmonella enterica serotype typhi
(S. typhi) or S. enterica serotype
paratyphi (S. paratyphi A,B,C) (Effa et al., 2011).
Food
products of animal origin are considered to be the major source of human Salmonella infection. Special programme
have been implemented for surveillance of poultry, swine and cattle. Also
include, the surveillance of healthy animals that maybe subclinical carriers of
Salmonella organisms. Cross–examination
during food processing is also monitored as contamination by healthy food
handlers can occur (World Health Organization (WHO), 1994).
Human
spreads Salmonella mainly through the
stool, food borne illness among the people and transmission can occur when food
and water are contaminated with stool or through direct fecal-oral route. Human
stool acts as an important reservoir of Salmonella
serovars that are the grouping of microorganisms based on their cell
surface antigen. Species isolated from human stool are Salmonella typhi, S. paratyphi
A, S. typhimurium, S. Wrothington and
S. enteritidis (Kumar et al.,
2009).
The
causative organism Salmonella typhi
has rapidly gained resistance of antibiotics like Ampicillin, Ceftriaxone and Co-trimazole
and also to previously efficacious drugs like Ciprofloxacin. The emergence of
antimicrobial resistance, especially the multidrug resistance to Ampicillin, Chloramphenicol,
and Co-trimazole, has further complicated the treatment and management of adult
and paediatic infections (Neopane, S. B Singh et al., 2008).
Antibiotic
sensitivity (susceptibility) is a term used to describe the susceptibility of
bacteria to antibiotics. The term “antibiotic” was derived from the ancient
Greek word “anti against and compound that kills or inhabits the growth of
bacteria (en-Wikipedia.org/wiki/antibiotic/sensitivity 2009). Antibiotic
susceptibility test is usually carried out to determine which antibiotic will
be most successful in treating a bacterial infection. The effectiveness of
individual antibiotics varies within location of the infection, the ability of
the antibiotic to resist or inactivate infection
(en-wikipedi.org/wiki/antibiotic sensitivity, 2009).
Antibiotic
sensitivity test is divided into three categories; susceptible, resistant and
intermediate categories. The “Susceptible” category implies that the isolates
are inhabited by the normal levels or concentration of antimicrobial agent
where the recommended dosage is used for treatment. The “intermediate” category
includes isolates with antimicrobial minimal inhibitory concentrations (MIC)
that approach usually attainable blood and tissue levels and for which response
rates may be used (e.g. beta-lactams). The “resistant” category implies that
isolates are not inhabited by the normal level or concentration of
antimicrobial zone diameter used fall in range where specific microbial resistance
(e.g. beta-lactams) are likely and the clinical efficacy of the agent against
the isolates has not being reliably show in treatment studies. (http://www.-uphs.upenn.edu/buddrug/antibioticmannual/amt.html,2009).
Treatment
with appropriate antibiotics is essential for recovery from typhoid fever. However,
treatment has come progressively more problematic with the gradual emergence of
antimicrobial resistance of Salmonella typhi.
Due to the resistance of S. typhi to antibiotics, there appears to
be the need to carry out antimicrobial test to check the susceptibility of this
organism to different antibiotics. The results from such anti-microbial test
would provide a good basis that will guide the choice of the moist effective
antibiotics that will be used in treating typhoid fever (Wain et al., 2001).
1.1
Aim
and Objectives
·
To isolate and characterize Salmonella typhi from stools of
symptomatic patients.
·
To investigate and check the
susceptibility pattern of Salmonella typhi
to selected routine antibiotics.
·
To recommend the antibiotics suitable
for use in Salmonella typhi from
stool samples.
1.2
Significance
of the Study
Knowing
the susceptibility of Salmonella typhi isolated from stool samples could
help in determining the routine antibiotics to be used on the patients.
CHAPTER
TWO
LITERATURE
REVIEW
2.1. The Organism
Typhoid
fever is caused by Salmonella typhi, a
gram-negative bacterium. A very similar but often less severe disease is caused
by Salmonella serotype paratyphi A. the nomenclature for these
bacteria is confused because the criteria for designating bacteria as
individual species are not clear. Two main views on the nomenclature of the
genus Salmonella have been discussed.
Le Minor and Popoff suggested that two species should be recognized. Salmonella bongori and Salmonella enterica. S. enteric include
six subspecies, of which subspecies I: (one) contained all the pathogens of warm–blooded
animals. Salmonella typhi was a
serotype within subspecies: Salmonella
enteric subspecies I serotype typhi (World
Health Organization (WHO), 2003).
Salmonella
typhi can only attack humans, so the
infection always comes from another human, either an ill person or a healthy
carrier of the bacterium. The bacterium is passed on with water and foods and
can withstand both drying and refrigeration (Charles, 2005).
In
the body, the organism can be found in the blood within the first 10 days of
infection from which it can be isolated. Later in the infection, it can be
found in stool and urine (Cheesebrough, 1993).
2.1.1 The Disease
During
an acute infection, Salmonella typhi multiplies
in mononuclear phagocytic cells before being released into the blooded stream. After
ingestion in food or water, typhoid organisms pass through the pylorus and reach
the small intestine. They rapidly penetrate the mucosal epithelium via either
microfold cells on enterocytes and arrive in the lamina propria, where they
rapidly elicit an influx of macrophages that ingests the bacilli, but does not
generally multiply. Some bacilli remain within macrophages of the small
intestinal lymphoid tissue. Other typhoid bacilli are drained into mesenteric
lymph nodes where there is further multiplication and ingestion by macrophages.
It is believed that typhoid bacilli reach the blood stream principally by lymph
drainage from mesenteric nodes, after which they enter the thoracic duct and
then the general circulation (World Health Organization, WHO, 2003).
2.1.2 Epidemiology
Typhoid
is transmitted mainly by the fecal-oral route, in most
cases an asymptomatic carrier of Salmonella
typhi or an individual who has
recently recovered from the infection continues to excrete large number of
organisms in the stool and contaminates food or water either through direct
food handling, through transfer of bacteria by flies and other insects or by
contamination potable water (Lin, et al.,
2000).
Approximately,
10% of patients recovering from typhoid fever excrete Salmonella typhi in the
stool for three months, and in the past 2-3% become permanent carries. These infections
have great potential for epidemic spread therefore (Spike, et al., 1987 and Thong, et al.,
1994). In the tropics enteric fever tends to be more common during the hot dry
seasons when the concentration of bacteria in rivers and streams increases, or
on the rainy season if flooding distributes sewage to drinking water sources.
In some areas the incidence of typhoid may be as high as 1, 000 cases per
100,000 population per year. In such areas typhoid is predominantly a disease
of children, and stool excretion of the infection. In such areas Salmonella typhi infections are commonly mild and self-limiting. Server
disease represents the “tip of the iceberg”. In temperate countries persistent
caries are more important reservoir of infection (Hoffman, et al., 1984).
For
travellers the highest attack rate are associated with visits to per (17 per 105
visits) J, India (11 per 105 visits J and Pakistan (10 per 10S
visits J. Although Indonesia has a reported annual incidence up to 1%, the
attack rate for travellers is low. In general, the mortality of enteric fever
is low (<10%) where antibiotics are available, but in poorer areas, or in
the context of natural disasters, wars, migrations large concentrated refuge
population and other privations, the mortality may rise to 10% - 30%, despite
antibiotic therapy. Typhoid tends to cluster in families (Luxemburger, et al.,
2001), presumably reflecting a common source of the infection and is associated
with poverty and poor housing. A part from explosive to the contaminated food
(often ice creams or iced drinks) or water source, a number of host factors
increase the risk of Salmonella
infections. Disease related (achlorhydria) or iatrogenic (antacids, H2
blockers, proton pump inhibitors) reduction in stomach acidity or get pathology
(surgery, inflammatory bowel disease, malignancy) and recent antibiotics
increase the susceptibility to infection. Disease related or iatrogenic
immunosuppression and several other infections, notably schistosomiasis, malaria
(Lucy et al., 1998 and Luxemburger et al., 2001), risk of Salmonella infections. Typhoid is more
common and more severe at the extremes of age. Neonatal typhoid usually
acquired from the mother may follow a fulminant course often with meningitis, patients
with hemoglobinopathies, particularly sickle cell dieses are also at increased
risk. (Bhutta, 1996 and Butter et al., 1991)
2.1.3 Mode of Transmission and Incubation Period
Humans
are the only natural host and reservoir, the infection is transmitted by
ingestion of food or water contaminated with faeces. Ice cream is recognized as
a significant risk factor for the transmission of typhoid fever. Selfish taken
from contaminated water and raw fruit and vegetables fertilized with sewage,
have been sources of past outbreaks. The highest incidence occurs where water
supplies serving large population are contaminated with faeces. Epidemiological
dated suggest that water borne transmission of Salmonella typhi usually
involves small inoculate, whereas food born transmission is associated with large
inoculate and high attack rates over short period (Evanoff et al., 1980).
The incubation period is 10-20 days and depends among other things, on how
large a dose of bacteria has been taken in. In the mild disease, the bacterium
is eliminated very early in the course of the disease and there are perhaps
only mild symptoms. It is possible to become a healthy carrier of infection
(Charles, 2005).
Mbuh
(1997) has reported different incubation periods, ranging from 3 to 40 days
with mean of 5 to 14 days. Water borne infection has long incubation period,
milk infection shorter and food the shortest.
2.1.4 Clinical
Manifestations
The
clinical features or manifestations of enteric fever vary considerably between
different geographic regions. In many areas typhoid becomes the leading
differential diagnosis of a patient with a fever which has lasted for more than
one week. The clinical features of typhoid and paratyphoid fever are generally
similar, although paratyphoid tends to be a more mild infection (Christie,
1984).
Most patients with enteric fever present with a
non-specific gradual onset of an influenza-like illness although Salmonella typhi infection can present with fever and a bewildering array of
signs and symptoms ranging from non-metastatic central nervous system syndromes
including psychosis and cerebellacadaxia (Trevett, et al., 1994), through to focal involvement of bone (Decleroq, et al., 1994) liver (El-Newihi, et al., 1996, Jagadish, et
al., 1994 and Schwartz, et al., 1994),
Spleen (Allal, et al, 1993), testes (Zafar, et
al., 1995), meninges (Lecour, et al.,
1994) ,and (Sharma, and Sharma, 1992), Vacular prostheses, antheromatous
plagues etc. (Van Basten, and Stockenbrugger, 1960).
In
general, the enteric fever is sub-acute infections with an incubation period of
approximately 7 – 14 days (range 3-6 days) following espouse. The illness begins
insidiously with non-specific signs and symptoms of fever (Pichons, et al., 1992), headache muscle and joint
aches, malaise, lassitude, anorexia, often a dry cough. (Sometimes associated
with a sore throat) (Sharma, and Sharma, 1992). The spleen enlarges, but
lymphaderiopathy is not usually prominent. There may be a few rose spots
(Sparse, pink, macular popular lesions which blanche with pressure and fade
after two or three days) on the thorax or abdomen (usually less than 10), but
these are often unnoticed (particularly in dark skinned patients). In
paratyphoid fever rose spots may be more prominent. The classic “step ladder
fever” of typhoid is unusual although the fever does become higher as the
disease progresses, until it levels fluctuating between 39oc and 40oc.
Mild chills and sweating are common but true rigors are rare. Relative bradycardia
is considered common in typhoid although in many series this has not been a
feature of the disease. Some abdominal complaints are usual although either
diarrhea or constipation may occur. There is usually some abdominal discomfort
and even in the first week of the disease the patient may notice passage
per-rectum of a small amount of blood or meleia. Normal bowel habit is usual in
typhoid. Diarrhea (Roy SK et al., 1985)
is more common in infants (Bhutta, et
al., 1991), and in patients with AIDS. Constipation occurs in approximately
40% of patients. A fulminant onset with a septic shock presentation may occur
bi is unusual. The clinical evolution of
untreated typhoid is divided classically into weeks (Christie, 1984).
During
the first week the fever rises gradually and in the second week reaches a high
plateau. By the second week the patient has become progressively weaker, has
lost weight and has often developed the characteristic affect from which
typhoid derives its name (typhoid means “like typhus”, which in turn drives
from the Greek typhos meaning smoke, and refers to the clouding of the
sensorium in these infection). The patient remains apathetic or depressed,
ahergic, often confused and withdrawn whilst lying in bed, yet sleep does not
come easily. By the third week of infection, if untreated, a dangerous stage is
entered upon in which either intestinal perforation or hemorrhage become more
likely as the necrotic peyeris patches either
erode through the wall of the terminal: leum (Bitar Rand Tarpley, 1985, Butter,
et al., 1991 and Butter, et al 1985),
or penetrate a large blood vessel. In large series reported before the
pre-chloramphenicol era intestinal hemorrhage occurred in 7 – 21% of cases and
intestinal perforation in between 0.7and 4.7% of cases (Christie, 1984).
In
the antibiotic era, the incidence of perforation has fallen slightly to
approximately 3% of cases, and clinically significant intestinal bleeding now
occurs in less than 2% although figures still vary considerably from series to
series. The risk of both hemorrhage and perforation increase from the middle of
the second week. In the third week of the illness, the patient is often
withdrawn, obtunded or intermittently delirious. The abdomen becomes distended
and there may be, vomiting and abdominal pain. Right upper quadrant pain may
indicate cholecystitis or cholangitis (35 of cases) whereas lower quadrant pain
with signs of peritoneal irritation may indicate perforation. Complications in
the third and fourth week also include preumonia (Parry, et al., 2002) ARDS (Buczko and Mclean, 1994), the development of
acute Psychosis, Coma (Rajeshwari et al.,
1995) and Biswa, 1994), myocarditis (Prabha, et al., 1995),
Pericarditis, orchitis (Zafar, et al., 1995),
Venous thrombosis (Ghosh and Samanta, 1994) Splenic rupture (Ali, et al., 1994), meningitis (Leocour, et al., 1994), hepatic dysfunction
(Jagadish et al., 1994) and
occasional renal failure (Shernurne, et
al., 2000). If the patient survive this phase of the illness there follows
a gradual recovery.
As
the duration of infection is an important determinate of the risk of severe
complications, a delay in receiving appropriate antibiotic treatment may have
serious consequences. In some endemic areas multi drug resistance (and thus
delayed treatment with effective antimicrobials) has led to an increase in
mortality, particularly in infants (Bhutta, 1996).
2.2 Diagnosis
The
clinical diagnosis of typhoid is confirmed by culture of the organism from
blood or born marrow or another non-gastrointestinal site. Isolation of the
organism from the duodenal secretions or the stool on a febrile patient is also
suggestive of enteric fever (although of course fever from a different
infection may occur in someone who has recently recoveredfrom typhoid or in a
chronic carrier). Stool cultures are positive in approximately 60% of children
and 25% of adults. 77 excretion of Salmonella
typhi in the stool is more likely
with higher blood bacterial counts and children tend to have higher bacteremia
than adults. Blood cultures are positive in 60% -80% of patients with yield maximized
by taking a large volume of blood. Lysis centrifugation and lysis plating
methods accelerate identification of Salmonella
typhi from blood (Saha, et al., 2001). The median bacterial
count is one cfu per ml of blood (Wain, et
al., 2001).
Approximately,
two thirds of these organisms are within phagocytic cells, and thus located in
the buffy coat (Rubin FA et al., 1990
and Wain j et al., 1998). Blood
bacterial counts decline as the disease progresses. Bone marrow counts are
approximately ten times higher (Wain j et
al., 2001). Bone marrow culture increases the diagnosis yield from blood
cultures by approximately 30% (Dance, et
al., 1991, Gilman, et al., 1975, Hoffman,
et al., 1986 and Hoffman, et al., 1984) Biopsies of the
rose-sports are also usually culture positive, Salmonella typhi is
usually present in the duodenum and can be recovered using the sting test
(Hoffman, et al., 1984). This is also
useful for identifying chronic gallbladder carriers (Gilman, et al., 1979). Salmonella typhi is
sometimes excreted in the urine and occasionally causes urinary tract
infections, particularly if there are structural abnormalities of the urinary
tract (Matthias, et al., 1995). In
areas where Schistosoma haematobiumis
endemic, such as Egypt, urinary tract carriers outnumber enteric carriers of Salmonella typhi. Urine antigen tests have been described recently (Chaicumpa
W et al., 1992) but these have not
been evaluated sufficiently several PCR methods have not been described, but
these are not used widely (Sharma, et
al., 1992), although molecular typins is important in distinguishing recrudescent
from newly acquired infections in endemic areas (Wain, et al., 1999) Serological diagnosis is widely relied upon (House, et al., 2001), the widal test which
measures the antibody titres to the somatic O and flagella H antigens is relied
upon widely, although there are very divergent views on its utility. There have
been several well documented epidemics in which the widal was usually negative,
but other epidemiological settings where it has proved useful. Overall
sensitivity is approximately 70-80% with specificity ranging from 80-95% (Parry,
et al., 1999), new Ig M and Ig G
based rapid serological tests have proved useful in some areas. (Bhutta, and
Mansurali, 1999; Choo, et al., 1999
and Lim, et al., 1998) but are not
validated sufficiently for widespread adoption.
2.3 Treatment
2.3.1
General Management
Supportive
measures are important in the management of Salmonella
typhi infection (typhoid fever), such
as oral or intravenous hydration, the use of antipyretic and appropriate
nutrition and blood transfusion if indicated. More than 90% of patients can be managed
at home with oral antibiotics, reliable care and close medical follow-up for
complication or failure to respond to therapy (Punjabi, 2000). However,
patients with persistent vomiting, severe diarrhea and abdominal distension may
require hospitalization and parenteral antibiotic therapy.
2.3.2 Antimicrobial Therapy
The
majority of patients with typhoid fever have uncomplicated infection and
present with a febrile illness. Most patients can take oral treatment and 90%
of cases are treated as outpatients. The proportion of patients who present
with complication infections varies considerably; for example in Nepal and Vietnam
the majority of patients with typhoid have uncomplicated infections and many
are still able to walk despite having positive blood cultures. In contrast, in
Jakarta, Indonesia, severe infections are relatively common, particularly in children
and are often present with encephalopathy or shock (Hoffman, et al., 1986). These patients obviously
require parental treatment. With the extensive spread of multidrug resistant Salmonella typhi chloramphenicol can no longer be regarded the drug of choice
for suspected enteric fever (Gulatis, et
al., 1992 and Rowe, et al., 1987).
In
complicated infections the median time to fever clearance in fully sensitive
infection is usually 4-5 days. It is not uncommon for blood cultures to remain
positive up to the third day of treatment with chloramphenicol in a fully
sensitive infection. The fluoroquinohone should now be regarded as the treatment
of first choice for typhoid fever. They sterilize the blood more rapidly than
other drugs and in general fever clearance times have ranged between 3 and 5
days within this group of drugs fever lasting one week following fluoroquinolone
treatment unusual and usually indicates halidixic-acid (i.e. quinolones)
resistance. With the third generation cephalosporius fever clearance times have
been longer, generally ranging between 5 and 8 days (Girgis, et al., 1995, Hien, et al., 1994, Smith, et al., 1994
and Wallace, et al., 1993). Recent
studies with short course fluoroquinolone treatment suggest that the duration
of fever does not reliably reflect the duration of infection. Two days treatment
with fluoroquinohone, which have over 85% treatment efficacy (Vinh, et al., 1996), are associated with a
fever clearance time of 4-5 days that
is, the patient is febrile for a longer period than the treatment is given. Patients
with background immunity will have a better response to treatment than non-immunes
and may self cure with ineffective drug treatment. Oligosymptomatic patient
with mild fever will usually respond rapidly and have a low incidence of
relapse. In recent study from Nepal high plasma concentrations of pro-inflammatory
cytokines, reflecting a more server infection, were associated with a delayed
response to antimicrobial treatment and an increased risk of relapse (Butter, et al., 1993).
Quinolone
resistance poses a major therapeutic problem; MDR quinolone resistant Salmonella typhi still usually responds to azithromycin, long courses of high
dose fluoroquinolones, or third generation cephalosporins, nut the optimum
treatment has not been determined.
2.3.4 Antimicrobial Susceptibility
Antimicrobial
susceptibility testing is crucial for the guidance of clinical management.
Isolates from many parts of the world are now multidrug-resistant (MDR) (Rome B
et al., 1997, Bhutta ZA, 1996 and
Gupta A, 1994). Isolates are usually resistant to ampicillin, chloramphenicol,
sulfonamide, trimethoprim, streptomycin and tetracycline. Alternative drugs
that are used for treatment include: Fluoroquinolones (e.g. Ceftriaxone), third
generation cephalosporin (e.g. Ceftriaxone, Cefotaxime) a mono bantam
beta-lactam (aztreonam) and a macrolide (azithromycin). Even though resistance
to the first two has been noted they nevertheless, remain useful (Saha, et al., 1999). Reduced susceptibility to
Fluoroquinolones is indicated by in vitro resistance to halidixic acid
(Murdoch, et al., 1998).
In
vitro susceptibility testing usually involves disc diffusion; the choice of
antimicrobial agents for the test is dictated by the agents that are currently
being used for treatment and the desire to determine the prevalence of MDR
stains. After the previous first-line drugs were discontinued for the treatment
of typhoid fever in Bangladesh because of the emergence of MDR strains, the
prevalence of multidrug resistance decreased and the possibility arose of using
these drugs again. It is therefore recommended that susceptibility tests be
performed against the following antimicrobial agents: a fluonquinolone, a third-generation cephalosporin
and any other drug currently used for treatment, halidixic-acid (for
determining reduced susceptibility to fluonquinolone because of the possibility
of false in vitro susceptibility against the fluonquinolone used for treatment)
and the previous first-line antimicrobials to which the stains could be
resistant (chloramphenol, ampicillin, trimethoprim/ sulfamethoxazole,
streptomycin and tetracycline). Azithromycin disc test result should be
interpreted with caution. The appropriate break-point recommendations for
azithromycin against Salmonella typhi are still not clear. Patients may
respond satisfactorily to azithromycin even if isolates are intermediate according
to current guidelines (Murdoch, et al., 1998).
CHAPTER
THREE
3.0 MATERIALS AND METHODS
3.1 Study Area
The
study area was Yusuf Dantsoho Memorial Hospital which is located in the
Northern part of Nigeria. The study was situated at 10.52 north latitude, 7.41
east longitude with about 1,582102 inhabitants residing in the state.
3.2 Sample Collection
A
total number of 100 samples of Salmonella
typhi of stool samples was collected from patients suffering from suspected
typhoid fever who attends Yusuf Dantsoho Memorial Hospital, Kaduna and put into
stool sample containers.
3.3 Methodology
3.3.1 Media Preparation
3.3.1.1
Macconkey Agar
52g
of Macconkey agar powder was dissolved in 1 litre of distilled water, the
mixture was then stirred properly to dissolve the agar. It was sterilized by
autoclaving at 1210c for 15 minutes and the medium was poured
aseptically in sterile petri dishes and allowed to solidify.
3.2.1.2
Selenite – F Broth
4g
of sodium biselenite was dissolved in 1 litre of distilled water, 19g of broth
base (1ab4a) was added and the both components was heated gently until it was
dissolved. It was allowed to cool rapidly, dispensed into final containers and
sterilized for 10minutes in a boiling water bath.
3.3.1.3
Salmonella-Shigella Agar
66g
of Salmonella – shigella agar was
dissolved in 1 litre of distilled water, where the mixture was boiled with
frequent agitation until the agar dissolved properly. It was allowed to cool to
500c then mixed and poured into sterile petri dishes to be
solidified.
3.3.1.4
Nutrient Agar
28g
of nutrient agar was dissolved in 1 litre of distilled water, it was allowed to
dissolve gently by autoclaving at 1210c for 15minutes. The medium
was poured aseptically in sterile petri dishes and allowed to solidify.
3.4 Isolation
A
loopful of faeces was inoculated on selenite – f broth alongside on Maconkey
agar and incubated at 370c for 24 hours then colourless colonies
which developed was sub-cultured on Salmonella
– shigella agar for 24 hours at 370c.
3.5 Gram Staining
·
Thin smears was prepared by using a
sterile wireloop to pick a small amount of colony from the discrete colonies
·
It was emulsified in small amount of
water on a sterile grease tree glass slide
·
The smear was allowed to dry then it was
heat fixed
·
The smear was then covered with crystal
violet for about 1 minutes
·
It was washed with water and covered
with grams iodine about for 1 minute
·
The slide was washed with water and
decolorized with alcohol for 10 – 30 seconds
·
The slide was washed with water and
finally covered with safranin stain for 10 – 30 seconds
·
It was washed with water and allowed to
dry
·
The back of the slide was wiped clean
and placed on a draining rack for the smear to air – dry
·
The slides was viewed using a microscope
with x100 objective lens
·
A pink (red) colour bacilli was observed
on the glass slide.
3.6 Antibiotic Sensitivity Disc testing
3.5g
of nutrient agar was dispensed into a sterile conical flask and 125ml of
distilled water was poured into the flask and stirred to dissolve the agar. The
mixture was autoclaved and poured into petri dishes. A colony was picked from
the isolates and spread on the plate containing nutrient agar. The gram
negative antibiotic sensitivity disc was introduced using sterile forceps and
then incubated for 24 hours at 370c. The antibiotics which were
susceptible or resistant and were recorded.
3.7 Biochemical characteristion and
identification
3.7.1 Catalase Test
A
drop of 3% hydrogen peroxide was placed on a clean glass slide and a colony of Salmonella isolate was picked with a
sterile wireloop and emulsified. The presence of bubbling and frothing was
observed which indicates a positive reaction.
3.7.2 Indole Test
Each
isolate was grown in 5mls of peptone water broth in a test tube for 24hours and
then 2 drops of kovec’s reagent was added, the mixture was carefully agitated.
A positive test is indicated by the development of a red colour in the reagent
layers above the broth within 1 minute. A negative test is indicated by the
reagent retaining its original yellow colour.
3.6.3 Oxidase Test
Two
drops of freshy prepared oxidase reagent was placed on a filter paper. A part
of the colony of the bacterial isolate was collected using one end of sterile
grease free glass slide and smeared across the filter paper impregnated with
the oxidase reagent and observed for deep purple colour within 10 seconds.
3.6.4 Motility Test
A
molten nutrient agar medium was poured in a sterile test tube and kept to
solidify. A growth will be picked from the isolates and inoculated into the
test tube by stabbing the medium with the aid of a straight wireloop and
incubated at 370c for 24hous. A diffused cloud extending away from
the line of inoculation will indicate a positive motility test. If the growth
is restricted to the line of inoculation and it is sharply defined which will
make the rest of the medium to be clear and indicates a negative motility test.
CHAPTER
FOUR
4.0 Results
The
work on the susceptibility of Salmonella typhis
isolated from stool samples of symptomatic patents attending Yusuf Dantsoho
Memorial hospital, Kaduna to selected routine antibiotics have been carried out
and the results are presented below. Out of the total of 100 samples subjected
to an analysis for Salmonella typhi,
13 (13%) isolates were identified by biochemical tests and the result is
presented on table 1.
From
the 13 (13%) Salmonella isolation 7
(53.84%) were Salmonella typhi and 6
(46.16%) were Salmonella typhimurium (Table
2) of the 13 Salmonella isolation 5
(38.46%) were from males and 8 (61.54%) from females.
Drug
susceptibility of the Salmonella Species
to ten (10) commonly used antibiotics in treating typhoid fever infection
showed that Salmonella typhi strains
showed resistance to Augmentin (25-ug), Septrin (30ug), Chloramphenicol (30ug)
and Amoxicillin (30ug) but were sensitive to ciprofloxacin (10ug), Gentamycin
(10ug), Pefloxacin (10ug), Tarivid (30ug), Streptomycin (30ug), and
Sparfloxacin (10ug) with mean zone of inhibitors of 21mm, 16mm, 20mm, 17mm,
21mm, and 19mm respectively.
Salmonella typhimurium
showed resistance to Augmentin (25ug) Septrin (30ug), Chloramphenicol (30ug)
and Amoxicillin (30ug) but were sensitive to ciprofloxacin (10ug), Gentamycin
(10ug), Pefloxacin (10ug), tarivid (30ug), Streptomycin (30ug), and
Sparfloxacin (10ug) with mean zone of inhibition of 19mm, 17mm, 22mm, 18mm, and
16mm, respectively (Table). The percentage resistance of the isolates against
the antibiotics used is 10% since the isolates were resistant to (4) four out
of (10) ten antibiotics used and the
percentage susceptibility of the isolates is 60% since the isolates were
fully susceptible to (6) six of the antibiotics used.
Table 1 Incidence of Salmonella Species
|
Total no of stool samples
|
No of males
|
No of females
|
No of Salmonella isolates
|
|
|
|
|
|
|
100
|
58
|
42
|
13
|
Table 2 Insistence and Sex
Distribution of Salmonella Species
Isolated From Stool Samples
|
Salmonella species
|
Number
of isolates
|
%
distribution according to sex
|
|
|
male %
|
Female
%
|
||
|
Salmonella
typhi
|
7 (53.84%)
|
2 (15.38%)
|
5 (38.46%)
|
|
Salmonella
Typhimurium
|
6 (46.16%)
|
3 (23.08%)
|
3 (23.08%)
|
|
|
13 (100%)
|
5 (38.46%)
|
8 (61.54%)
|
Table 3 Antibiotic Susceptibility
of Salmonella Species
|
Antibiotics used
|
Sensitive/resistant/ intermediate
|
Mean zone of inhibition (mm)
|
|||
|
Salmonella
typhi
|
Salmonella
typhimurium
|
Salmonella
typhi
|
Salmonella
typhimurium
|
||
|
Augmenting
(25ug)
|
R
|
R
|
-
|
-
|
|
|
Gentamycin
(10ug)
|
S
|
S
|
16
|
17
|
|
|
Pefloxacin
(10ug)
|
S
|
S
|
20
|
22
|
|
|
Tarivid (30ug)
|
S
|
S
|
17
|
18
|
|
|
Streptomycin
(30ug)
|
S
|
S
|
21
|
16
|
|
|
Septrin (30ug)
|
R
|
R
|
-
|
-
|
|
|
Chloramphenicol
(30ug)
|
R
|
R
|
-
|
-
|
|
|
Sparfloxacin
(10ug)
|
S
|
S
|
19
|
16
|
|
|
Ciprofloxacin
(10ug)
|
S
|
S
|
21
|
19
|
|
|
Amoxicillin
(30ug)
|
R
|
R
|
-
|
-
|
|
Keys: R-Resistant, S-Sensitive,
Ug-microgram, mm-millimeter
Biochemical
Tests
|
Organisms
|
Colony
morphology
|
Gram
charactenstics
|
Catalase
|
Indole
|
Oxidase
|
Motility
|
|
Salmonella
typhi
|
Single-milky colonies with black
dots
|
Gram-negative single rods
(Bacilli)
|
+
|
-
|
-
|
+
|
|
Salmonella
typhimurium
|
Single-mikly colonies with large amount of
blacks dots
|
Gram-negative single rods
(Bacilli)
|
+
|
-
|
-
|
+
|
Key + - positive
- - negative
CHAPTER
FIVE
5.0 Discussion
The
incidence of 13 positive results in 100 samples (Table 1) is of great public
heath concern, because these 13 people may serve as source of infection
resulting in the continuous spread of the infection unless they are properly
treated with the right antibiotic drug to cure the infection.
In
a study conducted in Yola, Adamawa state, to determine the resistance pattern
of Salmonella typhi to commonly used
antibiotics, 744 isolates were obtained from 974 samples from four different
hospitals in Yola between 2001-2004. This incidences however does reflect the
poor level of hygiene and water treatment being practiced today in Nigeria.
Otegbaya, (2003) reported that the availability and portability of drinking
water is still a luxury in Nigeria at the peak of the dry season especially in
Nigeria, water is often sourced from various doubtful places most of which are
contaminated by human waste.
Over-packed
refuse dump, as well as blocked and eutrophicating drainages with offensive
emissions of human and animals excreta are common sites in many parts of the
state, Consequently food and drinking water sources alongside broken pipes are
often easily contaminated by flies, cockroaches and rodents that scavenge for
food in these, contaminated environments resulting in high Salmonella infection rate.
In
this study, more females (61.54%) then males (38.46%) were infected, (Table 2),
However, both genders are equally susceptible to Salmonella infections.
The
demonstration of high rate of resistance by the isolates against Chloramphenicol,
Amoxicillin, Septrin, and Augmentin (Table 3) is a cause for great concern.
This is because these antibiotics which are usually the first line treatment of
typhoid fever can no longer be used for treating typhoid fever in this part of
the world. The result is in accordance with Mirza et al, (1996), which states that towards the end of 1980 and 1990 Salmonella typhi developed resistance
simultaneously to all drugs that were then used as first line treatment. In the
study, the isolates showed high susceptibility to Streptomycin Ciprofloxacin, Pefloxacin
Sparfloxacin Gentamycin and Tarivid (Table 4).
Uzma
et al, (2002) reported that there is
strong evidence that the fluoroquinolones are the most effective drugs for the
treatment of typhoid fever and other Salmonella
infections. The variation in the sensitivity pattern and high resistant
rates to these commonly used drugs could be attributed to the prevailing usage
and abuse and the common attitude of over the counter purchase of drugs in
Nigeria. Resistance by microorganisms to antibiotics may be an indication of
the presence of resistance factors such as B-lactamases and of recent extended
B-lactamases (EBL). The prevalence of (EBL) extended B-lactamses enzymes has
been increasing in many parts of the world. Infections caused by extended
B-lactamases producing isolates are difficult to treat, because they counter
resistance to all currently available B-lactam agents except Impipenem and in
some cases Piper acillintazobactam (Jones, 2001)
5.1 Conclusion
From
the result of this research, it can be seen that the incidence of Salmonella typhi (13%) during the study
period is of public heath importance and the antibiotic resistance (40%) among Salmonella typhi is common and
significant.
The
result also shows that only the Fluoroquinolones (Ciprofloxacin, Pefloxacin,
Sparfloxacin, Streptomycin, Gentamycin, and Tarivid) will effectively treat
typhoid fever and other Salmonella infections,
due to the (60%) susceptibility of the organism to these antibiotic used.
5.2 Recommendations
From
the study carried out it was discovered that the incidence of Salmonella infection was significant and
the organisms showed multi drug resistance to the routine antibiotics used
against them which makes treating typhoid fever and other Salmonella infections difficult. Therefore it is recommended that
1.
Specific days should be declared in a
month for environmental sanitation by the government.
2.
Vaccine for treatment of typhoid should
be acquired by the government to curb the incidence of typhoid in the country.
3.
Adequate training and facilities for
performing standardized sensitivity tests should be emphasized.
4.
Patients should be sufficiently informed
about isolation and their use to avoid abuse of antibiotics.
5.
Adequate laboratory facilities should be
provided for accurate diagnosis and oblation of pathogens to prevent the
inappropriate use of antibiotics.
6.
Cultural methods should be used as the
standard diagnostic method for typhoid fever so as to ensure that
susceptibility test is carried out on all suspected isolates before treatment
to select the most effective antimicrobial drug, instead of the use of widal
test, because the sensitivity, specificity and predictive values of this test
varies considerably among geographical regions.
7.
Antimicrobials should only be obtained
from recognized treatment centers and should not be taken without medical
supervision.
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