ANTIBIOGRAM STUDIES OF SALMONELLA ENTERITIDIS PHAGE TYPE 4 ISOLATES FROM POULTRY AND MEAT

Nusrat Shaheen, Naheed Fatima^*, Saraj-ud-Din Sajid^**, Abdus-Salam Khan Gandapur

Woman Medical College Abbottabad, ^*Department of Biological Sciences Quaid-i -Azam University Islamabad, ^**Frontier Medical College Abbottabad.

Background: Human infection with Salmonella enteritidis phage type 4 has increased worldwide since last decade and has been shown to be related mainly with the consumption of poultry meat and eggs. The public health significance and economic importance of this serovar underscores the need to generate base line data on the antimicrobial susceptibilities and protein profile of indigenous S. enteritidis. This study was performed to investigate the antimicrobial susceptibilities of S. enteritidis PhageType4 isolates from poultry and meat. Method: This study was carried out in the department of biological sciences Quaid-i-Azam University Islamabad during 1998-2000. A total of nineteen quinolone sensitive isolates of S. enteritidis from poultry meat and eggs collected during1994-1998 were characterized. The isolates were serotyped and phage typed at federal institute of consumer Health and Veterinary Medicine, Wernigerode Germany. Antimicrobial susceptibility tests were performed in accordance to the method of Bauer et al. (1966). Results: The results of the standard disc diffusion test showed 100% resistance against bacitracin, erythromycin and novobiocin. All (100%) isolates were highly sensitive to chloramphenicol. The results of minimum inhibitory concentration (MICs) tests using serial dilution of antimicrobial drugs revealed that 100% of the isolates were resistant against bacitracin, erythromycin and novobiocin at various levels of concentrations. Kanamycin, streptomycin and spectinomycin, all had very poor activity against serovar Enteritidis. Conclusion: These findings suggest the limited therapeutic potential and low typability of this serovar.

Key Words: Antibiogram, Salmonella Enteritidis, Phage Type 4.

INTRODUCTION

Non-typhoidal salmonellosis is a foodborne disease of primary concern in developed as well as developing countries. The spread of this disease is favored by wide array of animal reservoir and by the wide commercial distribution of both animals and food products and is one of the major public health problems in terms of socio-economic impact¹

Global surveillance data indicates that incidence of gastrointestinal infections caused by S. enteritidis has increased massively during the last decades. Among the S.enteritidis isolates, those assigned to phage type 4 (PT4) and phage type 8 (PT8) have been isolated predominantly from poultry and are the most frequent cause of human salmonellosis. Similar increase in S. enteritidis infections have also been reported in Europe, where phage type 4 has emerged as the predominant phage type, spreading rapidly through both poultry and human populations and virtually replacing all other phage types.² S. enteritidis has been reported to be responsible for 380 salmonellosis out- breaks in USA between 1985 and 1991, involving 13056 illnesses and 50 deaths.³

The most prevalent phage type isolated from humans in Europe is PT4, while the most common PTs isolated in North America are PT8 and PT13a.⁴ According to Rubino et al the most prevalent phage types present in S. typhimurium strains isolated in 1994 were DT104 followed by DT’s 124, 173, 193, 135, 12 and 99.⁵ Terajima et al reported the distribution of phage type of S. enteritidis isolates associated with the outbreak from human source in Japan during the year 1994 and 1995.⁶ PT1 and PT4 predominated with prevalence of 43.2% and 32.6% for 1994 and 35.3% and 31.8% for 1995, respectively. Ten different PTs were found among the 302 isolates phage typed PT4 was the predominant PT in both human (73.9%) and poultry (76.2%) isolates, followed by PT1 (8.0%), 8 (3.6%) and 7a (2.2%) in human isolates and by PT7a (4.9%), 1 (3.7%) and 12 (2.4%) in poultry isolates.⁷

Salmonella is an important food and water-borne pathogen around the world. It causes acute gastrointestinal illness. The infective dose can be as low as 15-20 cells.⁸

Despite improved sanitation, human infections with S. enteritidis have been increased worldwide since 1980 and shown to be related mainly to the consumption of poultry meat and eggs.⁷

S. enteritidis has been shown to cause gastroenteritis and other acute infections. There is however, little information on the antimicrobial susceptibilities and epidemiology of indigenous S. enteritidis PT4 isolates, which would help to prevent the spread of infections and provide data about the best choice for treatment. Our objectives in the present study were to investigate the antimicrobial susceptibilities of this organism. The study will have therapeutic, epidemiological and economic implications.

MATERIAL AND METHODS

This study was carried out in the department of biological sciences Quaid-i-Azam University Islamabad during 1998-2000. A total of nineteen quinolone sensitive isolates of. S enteritidis phage type 4 collected during 1994-1998 were used in this study. The isolates were serotyped and phage typed at the Federal Institute of Consumer Health and Veterinary Medicine, Wernigerode Germany.

Chemicals used in this study were obtained from Sigma and E. Merck and were of molecular biology grade. Culture media were purchased from Oxide Ltd and Difco Laboratories.

Antimicrobial susceptibility tests were performed in accordance to the method of Bauer et al. (1966).⁹ The Mueller Hinton Agar was used as growth medium for standard disc diffusion test. Staphylococcus. aureus ATCC 25923 strain was used in each antimicrobial assay to serve as control organism. Plates were incubated over night at 37^oC to check the sterility. Later on, 100 ml of test and control culture grown overnight in L.B broth at 37^oC were spread on plates with the heat sterilized glass spreader to form a smooth bacterial lawn. Standard susceptibility test disc impregnated with known agent and strength were dispensed on the agar surface. Within 15 minutes of application of the discs, plates were incubated overnight at 35^oC within 15 minutes after applying the disc. Characterization of strains as sensitive or resistant was based on the size of inhibition zone around the disc compared with the interpretation standards provided by the manufacturers. An isolate was defined as resistant, if it was resistant to at least one of the tested antimicrobial agent.

The antimicrobial drugs were dissolved (100mg/10ml) in the different volumes of water and ethanol. Different dilutions of each drug (100mg/10ml, 50mg/10ml, 25mg/10ml, 12.5mg/10ml, 6.25mg/10ml, 3.125mg/10ml) were prepared. Mueller Hinton agar was used as growth media for all MICs testing. Plates were poured and incubated over night to check their sterility. 100ml of an over night culture was spread on the plates with heat sterilized glass spreader to form a smooth bacterial lawn. The sterilized blank discs were placed on the surface of the medium .The distance between the disc was kept approximately 2 cm. The 10ml of each antimicrobial drug dilution was poured per disc in descending order of concentration. The plates were inoculated over night at 37⁰C and the diameter (mm) of inhibition zones was recorded the next day. All solutions and dilutions of antimicrobials were made fresh and all handling was done using sterile equipment under sterile conditions.

RESULTS

The results of minimum inhibitory concentration (MICs), and antibiogram of different isolates are shown in tables 1 and 2 respectively.

Standard Disc Diffusion Tests Results

The results of the standard disc diffusion tests and antibiogram of different isolates are given in tables 3 and 4 respectively.

Table-1: Percentage resistance of 19 Salmonella enteritidis PT4 isolates against different concentrations of antimicrobial drugs

Antimicrobial drug concentration mg/discs
ANTIMICROBIAL DRUGS	100	50	25	12.5	6.25	3.125
AMPICILLIN	15.78% (3)	5.26% (1)	21.05% (4)	15.78% (3)	10.52% (2)	-
BACITRACIN	100% (19)	100% (19)	100% (19)	100% (19)	100% (19)	100% (19)
CHLORAMPHENICOL	-	-	-	-	5.26% (1)	36.84% (7)
ERYTHROMYCIN	100% (19)	100% (19)	100% (19)	100% (19)	100% (19)	100% (19)
GENTAMYCIN	-	5.26% (1)	10.52 (2)	5.26% (1)	31.57% (6)	31.57% (6)
KANAMYCIN	31.57% (6)	10.52% (2)	10.52% (2)	21.05% (4)	21.05% (4)	5.26% (1)
NOVOBIOCIN	100% (19)	100% (19)	100% (19)	100% (19)	100% (19)	100% (19)
PENICILLIN	26.31% (5)	10.52% (2)	21.05% (4)	21.05% (4)	10.52% (2)	-
SPECTINOMYCIN	68.42% (13)	21.05% (4)	5.26% (1)	-	5.26% (1)	-
STREPTOMYCIN	10.52% (2)	21.05% (4)	47.36% (9)	21.05% (4)	-	-
TETRACYCLINE	31.57% (6)	36.84% (7)	26.31% (5)	-	-	-
TRIMETHOPRIM	63.15% (12)	-	5.25% (1)	-	10.52% (2)	-

Note: No. of isolates resistant are given in the parenthesis.

Table-2: Antibiogram of individual isolate of Salmonella enteritidis PT4

ISOLATES NO	ANTIMICROBIAL DRUG RESISTANCE PHENOTYPE
S.E 1	KN^1, SP^1, ST^4, TP^1, TE^1, AP^5, PN^5, CM^6, GM^6, NB^1, EM^1, BC^1
S.E 2	NB^1, SP^2, AP^1, CM^5, KN^1, ST^4, TE^1, PN^1, TP^1, EM^1, BC^1
S.E 3	KN^1, SP^1, ST^2, TP^1, TE^1, AP^5, PN^3, CM^6, GM^6, NB^1, EM^1, BC^1
S.E.4	TE^1 , NB^1,KN^6, ST^1, SP^3, , BC^1, EM^1
S.E 5	AP^3, NB^1, SP^1, PN^1, ST^2, KN^5, TE^3, TP^5, GM^5, EM^1, BC^1
S.E.6	SP^1, KN^4, ST^3, TP^5, TE^3, AP^2, PN^3, CM^6, GM^5, NB^1, EM^1, BC^1
S.E 7	PN^4, SP^1, AP^3, GM^5, KN^2, ST^2, NB^1, TE^2, TP^1, EM^1, BC^1
S.E 8	TP^1, TE^2, NB^1, AP^4, KN^1, ST^1, PN^5, SP^5, EM^1, BC^1
S.E 9	PN^4, SP^1, AP^1, GM^3, KN^5, ST^3, NB^1, TE^2, TP^1, EM^1, BC^1
S.E10	AP^3, NB^1, SP^1, PN^1, ST^3, KN^4, TE^2, TP^1, GM^6, EM^1, BC^1
S.E11	SP^1, KN^3, ST^4, TP^1, TE^2, AP^3, PN^1, CM^6, GM^4, NB^1, EM^1, BC^1
S.E12	TE^2, ST^3, NB^1, KN^4, SP^1, PN^3, GM^2,EM^1, BC^1
S.E13	TP^1, TE^1, NB^1, GM^5, KN^2, ST^3, PN^3, SP^1, EM^1, BC^1
S.E14	ST^3, TE^3, NB^1, GM^5, KN^4, AP^4, PN^1, SP^2, EM^1, BC^1
S.E15	SP^1, KN^3, ST^3, TP^3, TE^1, AP^4, PN^2, CM^6, GM^3, NB^1, EM^1, BC^1
S.E16	NB^1, SP^1, GM^5, CM^6, KN^5, ST^2, TE^3, PN^4, TP^1, EM^1, BC^1
S.E17	TE^2, ST^3, NB^1, KN^5, SP^2, PN^4, GM^6, EM^1, BC^1
S.E18	ST^3, TE^3, NB^1, GM^6, KN^1, TP^1, PN^2, SP^2, EM^1, BC^1
S.E19	KN^1, SP^1, ST^3, TP^1, TE^1, AP^1, PN^4, CM^6, GM^6, NB^1, EM^1, BC^1

Note: The numbers 1, 2, 3, 4, 5 and 6 represent the antimicrobial drug concentrations (mg/disc) as follows:

1 = 100 mg/disc 2 =50 mg/disc 3 = 25 mg/disc 4 = 12.5 mg/disc 5 = 6.25 mg/disc 6 = 3.125 mg/disc

TABLE-3: Antimicrobial drug susceptibility testing of nineteen Salmonella enteritidis PT4 isolates using standard antimicrobial drug disc

ANTIMICROBIAL DRUGS	% RESISTANT	% INTERMEDIATE	% SUSCEPTIBLE
AMPICILLIN	-	5.26 % (1)	94.73 % (18)
BACITRACIN	100% (19)	-	-
CEFOPERAZONE	-	68.42% (13)	26.31 % (5)
CHLORAMPHENICOL	-	-	100% (19)
ERYTHROMYCIN	100 % (19)	-	-
FORTUM CEFTAZIDIME	42.10% (8)	5.26% (1)	31.57% (6)
GENTAMYCIN	78.94 % (15)	10.52 % (2)	5.26 % (1)
KANAMYCIN	42.10% (8)	42.10 % (8)	15.78% (3)
NOVOBIOCIN	100% (19)	-	-
PENICILLIN	89.47% (17)	-	10.25 % (2)
SXT*	52.63 % (10)	5.26% (1)	42.10 % (8)
STREPTOMYCIN	94.73% (18)	-	5.26% (1)
TRIMETHOPRIM	68.42 % (13)	-	26.31 % (5)
TETRACYCLINE	31.57 % (6)	5.26 % (1)	63.15 % (12)

Note: No. of isolates resistant are given in the parenthesis. SXT*(Septran Co Trimoxazole)

Table-4: Antibiogram of individual resistant isolates of Salmonella enteritidis PT4 for standard antimicrobial drugs discs

Isolate no:	Antimicrobial drug resistance phenotype
S.E 1	EM , PN , TE , KN , ST , NB , SX, TP , GM , BC
S.E 2	EM , PN , KN , SX , GM , CA , NB , TE , BC
S.E 3	EM , SX ,CA , GM , KN , ST , TE , NB , TP , BC
S.E 4	EM , SX , PN , ST , TE , NB , CA , BC
S.E 5	EM , SX ,CA , GM , PN , ST ,TE , NB , TP , BC
S.E 6	EM , TP , PN , ST , CA, SX, GM , KN , NB , BC
S.E 7	EM , CA , TP , KN, GM , PN , ST , TE , NB , SP , SX, BC
S.E 8	EM , SX, PN , ST , NB , TP , GM , BC
S.E 9	EM , CA, PN , ST , NB , SX , TP , GM , BC
S.E10	EM , PN , ST , CA , GM , NB , BC
S.E 11	EM , PN , ST , NB , KN , GM , BC
S.E 12	EM , PN , ST , NB , GM , BC
S.E 13	EM , PN , ST , NB , GM , BC
S.E 14	EM , PN , ST , GM , NB , BC
S.E 16	EM , PN , ST , TP , NB , GM , BC
S.E 17	EM , PN , ST , KN , NB , TP , BC
S.E 18	EM , NB , KN , TP, BC
S.E 19	EM , TP , TE , SX , PN , NB , TE , ST , KN , BC

*CAZ (Fortum Ceftazidime ) *SXT (Septran Co Trimoxazole )

DISCUSSION

For the antimicrobial drugs used against nineteen S. enteritidis isolates, the results of MICs showed nine different phenotypic patterns. The MICs data demonstrated very high level of resistance to erythromycin, bacitracin and novobiocin, as 100% of the isolates showed resistance at higher concentrations of these drugs (See table: 1). Previous studies on 86 strains of S. enteritidis, isolated from poultry and poultry environment by Singer et al. (1992) has also demonstrated 100% resistance to bacitracin.¹⁰The development and spread of antimicrobial resistance to bacitracin may be linked to selection pressure caused by excessive use of this drug.¹¹ Salmonella resistance at varying concentrations of penicillin, chloramphenicol, streptomycin, spectinomycin and erythromycin has also been reported by other workers.^12,13,14,15 The higher resistance rates of S. enteritidis to tetracycline, bacitracin and kanamycin mandate the consideration of other therapeutic options and suggest the limited use of therapeutic potentials of these antimicrobial agents.

Kanamycin has been previously recommended as drug of choice against Salmonella,¹⁶however, our studies as well as those of Mansoor, shows that kanamycin MICs for all of the Salmonella isolates are relatively higher.¹⁵ These finding indicates that kanamycin should not be recommended in our populations against Salmonella infection. The results of in vitro susceptibility by standard disc showed that all isolates were highly resistant to erythromycin (15mg/disc), novobiocin (5mg/disc) and bacitracin (10units/disc), followed by streptomycin (10mg/disc) and penicillin (10 units/disc) to which 94.73 % and 89.47 % isolates were resistant respectively (See table-1). The over all frequencies of resistance to gentamycin (10mg/disc), trimethoprim (5mg/disc) and ceftazidime (30mg/disc) were 78.9%, 68.4% and 42.10% respectively (See table:1). These levels of resistance are quite different than those reported in previous studies by Athar .¹⁷ During six years (1978-1982) study, 105 paratyphoid organisms belonging to 32 serovars were reported with sensitivity to tetracycline (10 units/disc) and six other antimicrobial drugs, including furazolidone, gentamycin, neomycin, penicillin and erythromycin. In another study, Javed (1992) demonstrated the susceptibility of various Salmonella serovars to several antimicrobial drugs using 112 Salmonella strains and reported high resistance to kanamycin followed by trimethoprim sulphamethoxazole and tetracyline.¹⁸ Similar trend to these antimicrobial drugs has also been reported by others .^19,20,21 The frequency of antimicrobial drugs resistance in S. enteritidis has been found to be low and stable.^{22, 23} The majority of the strain of S.enteritidis continue to be fully sensitive to antimicrobial drugs and of 18,968 isolates reported by Laboratory of Enteric Pathogen in 1996, only eight were resistant with less than 0.5% resistance to four or more drugs.²⁴ The antimicrobial susceptibility and molecular epidemiology of 275 S. enteritidis strains isolated in Hong Kong from 1986-1996 were studied. Over 99% of these isolates were susceptible to 17 of the 19 antimicrobials tested. One isolate harbored an autotransfering plasmid that confers resistance to tetracycline, trimethoprim-sulfamethoxazole. Another isolate harbored a mobilizable plasmid that confers resistance to ampicillin and cephalothin.²⁵

Antimicrobial resistance in Salmonella strains is generally encoded by plasmid, which has been acquired as consequence of antibiotic pressure in humans and veterinary medicine, however, due to the fluidity of resistant plasmids and transposons, antimicrobial drug resistance pattern can not be recorded as satisfactory method for discriminations within serovars.²⁶ This has also been observed in our study.

However antimicrobial resistance typing can be used in conjunction with serotyping, phage typing, protein analysis and genetic characterization of resistance plasmid for epidemiological purposes²⁷. Under such circumstances antibiogram should continuously be monitored to keep up to date with changes in drug resistance pattern.

In summary, both the results, i.e. standard disc and serial dilution MICs indicate the limited therapeutic value of bacitracin, erythromycin, kanamycin, streptomycin and spectinomycin (See table 1, 3) The need for continued surveillance is emphasized to determine local antimicrobial susceptibility data to identify changing pattern of resistance. Such data is essential for developing appropriate treatment of salmonellosis. Moreover, the prevalence of highly susceptible S. enteritidis PT4 strains suggest the limited use of antibiogram as an epidemiological marker as reported previously.²⁶

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Address for correspondence:

Nusrat Shaheen, Assistant Professor, Department of Biochemistry, Women Medical College Abbottabad, Pakistan.

Email: natrasweet@yahoo.com