Journal Of Clinical Microbiology 1999 Navia 3113.full [620749]

JOURNAL OF CLINICAL MICROBIOLOGY ,
0095-1137/99/$04.0010Oct. 1999, p. 3113–3117 Vol. 37, No. 10
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Typing and Characterization of Mechanisms of Resistance of
Shigella spp. Isolated from Feces of Children under
5 Years of Age from Ifakara, Tanzania
MARGARITA M. NAVIA,1LILIANA CAPITANO,1JOAQUIN RUIZ,1MARTHA VARGAS,1
HONORATI URASSA,2DAVID SCHELLEMBERG,3JOAQUIM GASCON,4AND JORDI VILA1*
Departament de Microbiologia,1Malaties Infeccioses (Unitat de Medicina Tropical),4and Unitat de
Epidemiologia i Bioestadistica (Fundacio ´ Clinic),3Hospital Clinic, Institut d’Investigacions
Biome `diques August Pı ´ i Sunyer, Villarroel 170, Barcelona 08036, Spain, and
Ifakara Health Research and Development Centre, National
Institute for Medical Research, Ifakara, Tanzania2
Received 19 March 1999/Returned for modification 29 April 1999/Accepted 29 June 1999
Eighty-six strains of Shigella spp. were isolated during the dry season from stool samples of children under
5 years of age in Ifakara, Tanzania. The epidemiological relationship as well as the antimicrobial susceptibility
and mechanisms of resistance to ampicillin, chloramphenicol, and co-trimoxazole were investigated. Fourdifferent epidemiological tools, pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic(REP)-PCR, plasmid analysis, and antibiogram, were compared for typing Shigella strains. Seventy-eight (90%)
strains were Shigella flexneri and were distributed into four groups, by either PFGE or REP-PCR, with 51, 17,
7, and 3 strains. The four strains of Shigella dysenteriae belonged to the same group, and the four strains of
Shigella sonnei were distributed in two groups with three and one strain each. Plasmid analysis showed a high
level of heterogeneity among strains belonging to the same PFGE group, while the antibiogram was lessdiscriminative. REP-PCR provided an alternative, rapid, powerful genotyping method for Shigella spp. Overall,
antimicrobial susceptibility testing showed a high level of resistance to ampicillin (81.8%), chloramphenicol(72.7%), tetracycline (96.9%), and co-trimoxazole (87.9%). Ampicillin resistance was related to an integron-borne OXA-1-type b-lactamase in 85.1% of the cases and to a TEM-1-type b-lactamase in the remaining 14.8%.
Resistance to co-trimoxazole was due to the presence of a dhfr Ia gene in all groups except one of S. flexneri,
where a dhfr VII gene was found within an integron. Chloramphenicol resistance was associated in every case
with positive chloramphenicol acetyltransferase activity. All strains were susceptible to nalidixic acid, cipro-floxacin, ceftazidime, cefotaxime, and cefoxitin. Therefore, these antimicrobial agents may be good alternativesfor the treatment of diarrhea caused by Shigella in Tanzania.
Acute infectious diarrheal disease is one of the most fre-
quent causes of childhood deaths in the developing world.Diarrheal disease accounts for approximately 25% of all deathsin children younger than 5 years of age in these areas (21).Infections caused by Shigella species are an important cause of
diarrheal disease, in both developing and developed countries.Worldwide, it is estimated that shigellosis causes around600,000 deaths per year, two-thirds of the deceased being chil-dren under 10 years of age. Shigella dysenteriae and Shigella
flexneri are the predominant species in the tropics, while Shi-
gella sonnei is the predominant species in industrialized coun-
tries (18).
Shigellosis is one of the acute diarrheal diseases for which
antimicrobial therapy is effective. However, today it also pre-sents a pressing challenge, as Shigella spp. have progressively
become resistant over the past decades to most of the widelyused and inexpensive antimicrobials (21). Thus, the history ofthe genus suggests that resistance will emerge to any antimi-crobial agent used intensively (25). Antimicrobial resistance inenteric pathogens is of the greatest importance in the devel-oping world, where the rate of diarrheal diseases is highest andindiscriminate use of antimicrobial agents is a fact.
The comparative analysis of different epidemiological mark-ers is important in order to know which is the best for tracingthe source of infection during an outbreak. Several conven-tional typing methods and newly introduced molecular biologytyping techniques have been described (3, 5, 11, 13). On theother hand, the study of the mechanisms of resistance ofthe resistant pathogenic bacteria may provide insight into themeans by which multiple resistance is spreading among thebacterial population.
The aim of this article is to characterize Shigella strains
isolated from children under 5 years of age in Ifakara, Tanza-nia. The work includes comparative epidemiological typingwith various epidemiological tools, as well as a determinationof antimicrobial susceptibility and the molecular characteriza-tion of the mechanisms of resistance to ampicillin, chloram-phenicol, and co-trimoxazole.
MATERIALS AND METHODS
Bacterial strains. Eighty-six strains of Shigella spp. were isolated from stool
samples of children under 5 years of age during the dry period (July to Septem-
ber) of 1997 in Ifakara, Tanzania. The children included in the study were seenat Saint Francis Designated District Hospital. Shigella spp. were identified by
conventional methods (16) and by serotyping. All the strains with differentplasmid patterns or antibiograms were investigated in detail to determine theirmechanisms of resistance to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole.
Antimicrobial susceptibility testing. Susceptibility testing was performed by an
agar diffusion disk method as recommended by the National Committee forClinical Laboratory Standards (17). Mueller-Hinton agar was obtained fromBecton Dickinson (Cockeysville, Md.), and antimicrobial disks were obtained
* Corresponding author. Mailing address: Laboratori de Microbio-
logia, Hospital Clinic, Villarroel 170, Barcelona 08036, Spain. Phone:
(34) 932275522. Fax: (34) 932275454. E-mail: vila@medicina.ub.es.
3113 on December 3, 2019 by guest http://jcm.asm.org/ Downloaded from

from BBL Microbiology Systems (Cockeysville, Md.). Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC
27853 were used as quality control organisms and tested weekly. Each time a new
batch of Mueller-Hinton agar was introduced, Enterococcus faecalis ATCC 29212
was tested to detect the presence of inhibitors of trimethoprim-sulfamethoxazole.The MICs of ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, tetracy-
cline, cefoxitin, cefotaxime, ceftazidime, nalidixic acid, and ciprofloxacin for theselected strains were determined by E-test strips (AB Biodisk, Solna, Sweden) onMueller-Hinton agar plates, following the manufacturer’s instructions. E. coli
ATCC 25922 was used as a reference strain for quality control.
Low-frequency restriction analysis of chromosomal DNA by PFGE. Genomic
DNA was prepared as described previously (15), digested with XbaI, and sepa-
rated in 1% agarose gels with a contour-clamped homogeneous-field apparatus(CHEF-DR2; Bio-Rad). It was run under 200 V, with the pulse time increasingfrom 5 to 8 for 20 h. Pulsed-field gel electrophoresis (PFGE) patterns wereinterpreted by using the criteria established by Tenover et al. (26).
REP-PCR. Repetitive extragenic palindromic (REP)-PCR was carried out
following the method previously described by Gallardo et al. (6), with somemodifications. Briefly, the primer 59 GCG CCG ICA TGC GGC ATT 39 was
used under the following conditions: 30 cycles of 1 min at 94°C, 1 min at 40°C,and 1 min at 65°C, with a final extension of 16 min at 65°C. The reaction wasprepared with 5 ml of boiled bacterial suspension, 1 ml of 5 mM primer, and PCR
beads (Pharmacia A.B., Uppsala, Sweden). Fifteen microliters of the PCR prod-ucts was separated in a 12.5% precast polyacrylamide gel with a Genephorapparatus (Pharmacia) and silver stained.
Plasmid analysis. Plasmid DNA was extracted from overnight bacterial cul-
tures with the commercial kit Wizard Plus SV Minipreps DNA purificationsystem (Promega, Madison, Wis.) according to the manufacturer’s instructions.The plasmids obtained were visualized and analyzed by 0.8% agarose gel elec-trophoresis.
b-Lactamase detection. b-Lactamase analysis was performed by the following
methods.
(i) Isoelectrofocusing. Isoelectrofocusing was performed as described else-
where (6). Gels were run in a PhastSystem apparatus (Pharmacia A.B.) anddeveloped with nitrocefin, and the isoelectric points were determined. Severalstrains carrying b-lactamases of known pI were used as controls and focused in
parallel with the extracts.
(ii) PCR. All PCR amplifications of the different b-lactamase genes were
carried out in a DNA Thermal Cycler 480 (Perkin-Elmer Cetus, Emeryville,Calif.), using the primers previously described (6) and under the followingconditions: 30 cycles of denaturation at 94°C, annealing at 55°C, and extension at72°C, plus a final extension of 7 min at 72°C. The PCR product was run andvisualized in 0.7% agarose gels stained with ethidium bromide.
Chloramphenicol acetyltransferase detection. The chloramphenicol acetyl-
transferase activity assay was performed as described elsewhere (2), with slightmodifications (6). Briefly, the strains were grown overnight on MacConkey agar.A heavy suspension of bacteria in 0.2 ml of 1 M NaCl, 0.01 M EDTA, and 0.05%sodium dodecyl sulfate (pH 8) was incubated in an Eppendorf tube at 37°C for60 min. After a short centrifugation in a microcentrifuge, 50 ml was transferred
to a microtitration plate. Duplicate wells were prepared with each strain, and 50ml of a solution containing two parts 0.2 M Tris-HCl (pH 8), 2 mM acetylcoenzyme A, and one part 10 mM 5,5-dithio-bis-(2-nitrobenzoic acid) in 0.1 MTris-HCl, pH 8, was added to each well. A 50- ml amount of 5 mM sterile
chloramphenicol (dissolved in water) was added to one well (test reaction), andan equivalent amount of water was added to the duplicate well (control). Theplate was reincubated at 37°C for 5 min. The reaction was stopped by adding 1NH
2SO4and read spectrophotometrically.
Detection of trimethoprim resistance genes. Both dhfr Ia and dhfr VII genes
were amplified under the same conditions used for b-lactamases and with the
following primers: dhfr Ia upper (59 GTG AAA CTA TCA CTA ATG G 39) and
lower (59 TTA ACC CTT TTG CCA GAT TT 39) and dhfr VII upper (59 TTGAAA ATT TCA TTG ATT G 39) and lower (59 TTA GCC TTT TTT CCA AAT
CT 39). The sizes of the PCR products for both genes were the same, 474 bp, andincluded the entire gene.
Integron amplification and cloning. Reaction mixtures for integron amplifi-
cation were prepared in the same way as those for b-lactamase PCR but with the
following primers: upper (59 GGC ATC CAA GCA GCA AG 39) and lower (59
AAG CAG ACT TGA CCT GA 39) (10). The conditions for amplification wereas follows: 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 8 min, plusa final extension of 72°C for 16 min. Twenty-five microliters of the amplifiedproducts was run in a 1.5% agarose gel and stained with ethidium bromide. Thebands were excised from the gel, and the DNA was recovered with a GeneCleankit (Bio 101, Inc., La Jolla, Calif.) and cloned into pCRII vector (Invitrogen BV,Leek, The Netherlands).
DNA sequencing. Plasmid extraction was performed as described above. The
sequencing of the plasmids with the cloned inserts was done with a Thermo-sequenase dye terminator sequencing kit in an automatic DNA sequencer (mod-el 377; Applied Biosystems, Perkin-Elmer, Emeryville, Calif.) following the manu-facturer’s instructions.
RESULTS
The eighty-six strains of Shigella spp. that were isolated were
distributed as follows: 78 (90%) were S. flexneri, 4 (4.6%) were
S. dysenteriae, and 4 (4.6%) were S. sonnei.N oShigella boydii
strains were isolated. The 78 S. flexneri strains were grouped
into four epidemiological groups by PFGE or REP-PCR (Fig.
1 and 2). The distribution of strains according to these epide-miological markers was as follows: 51 strains in group F-I, 17strains in group F-II, 7 strains in group F-III, and 3 strains ingroup F-IV. However, the four major S. flexneri groups were
subdivided into nine different subgroups based on antibiogramand plasmid analysis (Table 1). Eight different plasmid pat-terns were obtained among S. flexneri strains (Fig. 3). These
patterns contained from three to six different plasmids each,although in some cases the difference between two patternswas due to the gain or loss of only one plasmid.
On the basis of antibiotic susceptibility, six phenotypes were
defined: phenotype I (Amp
rCmrTetrSxtr), phenotype II
(AmpsCmsTetsSxts), phenotype III (AmpsCmsTetrSxts),
phenotype IV (AmpsCmsTetrSxtr), phenotype V (AmprCms
TetrSxtr), and phenotype VI (AmprCmrTetrSxts). In spite of
belonging to the same clone by PFGE, REP-PCR, or plasmidanalysis (Fig. 1 to 3), the four strains of S. sonnei were distrib-
FIG. 1. PFGE. Lanes 1, 2, and 3, S. flexneri strains belonging to group F-I;
lanes 4, 5, and 6, S. flexneri strains belonging to group F-II; lanes 7, 8, and 9, S.
flexneri strains belonging to group F-III; lanes 10 and 11, S. flexneri strains
belonging to group F-IV; lanes 12 and 13, S. dysenteriae strains; lanes 14 and 15,
S. sonnei strains.
FIG. 2. REP-PCR. Lanes A and P, molecular size markers; lanes B and C, S.
flexneri strains belonging to group F-II; lanes D, E, and F, S. flexneri strains
belonging to group F-I; lanes G and H, S. flexneri strains belonging to group
F-III; lanes I and J, S. flexneri strains belonging to group F-IV; lanes K, L, and
M,S. dysenteriae strains; lanes N and O, S. sonnei strains.3114 NAVIA ET AL. J. C LIN.M ICROBIOL . on December 3, 2019 by guest http://jcm.asm.org/ Downloaded from

uted in two groups based on the antibiogram. Three strains
showed phenotype IV (group S1), and one strain showed phe-notype V (group S2). The four strains of S. dysenteriae were all
the same clone (Table 1).
Fourteen S. flexneri strains, three S. sonnei strains, and three
S. dysenteriae strains were used for detailed investigations of
the mechanisms of resistance to ampicillin, chloramphenicol,and co-trimoxazole. The MICs of ampicillin, chloramphenicol,tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin, cefta-zidime, cefotaxime, and cefoxitin for these strains are shown inTable 2. For all the strains resistant to ampicillin, chloram-phenicol, and tetracycline, the MICs of the drugs were .256
mg/ml, and for those resistant to co-trimoxazole, the MIC was.32 mg/ml. Overall, 92% of S. flexneri strains were resistant to
ampicillin and chloramphenicol, 99% were resistant to tetra-cycline, and 91% were resistant to co-trimoxazole. S. dysente-
riae strains were 100% resistant to ampicillin, chlorampheni-
col, tetracycline, and co-trimoxazole. While S. sonnei strains
were all susceptible to chloramphenicol, only one of fourstrains was resistant to ampicillin and all showed resistance totetracycline and co-trimoxazole. All Shigella sp. strains tested
were susceptible to nalidixic acid, ciprofloxacin, cefotaxime,ceftazidime, and cefoxitin (Table 2).
Isoelectric focusing was used first to detect the production of
b-lactamase, and PCR with specific primers was used to cor-
roborate the results, which are shown in Table 3. The ampicil-lin resistance of S. flexneri was explained in 75% of the cases by
the presence of an OXA-1-type b-lactamase (pI 7.0), whereas
the remaining 25% had a TEM-1-type b-lactamase (pI 5.4).
The S. dysenteriae clone also carried an OXA-1-type b-lactam-
ase, whereas the ampicillin-resistant S. sonnei strain had a
TEM-1-type b-lactamase. In all cases, the OXA-1-type b-lac-
tamase was located in an integron (data not shown).
All strains resistant to chloramphenicol showed chloramphen-
icol acetyltransferase activity (Table 3), and also, all co-trimox-
azole-resistant strains presented genes encoding dihydrofolatereductases (Table 3). Four of five co-trimoxazole-resistantS. flexneri epidemiological groups showed the dhfr Ia gene, and
the fifth group showed a dhfr VII gene, while co-trimoxazole-
resistant S. sonnei and S. dysenteriae strains also had the dhfr
Iagene.DISCUSSION
The predominant species of Shigella during the studied pe-
riod of time was S. flexneri, which is usually the predominant
species in areas of endemicity, accounting for 50% of culture-
positive disease (25). S. sonnei andS. dysenteriae were found in
the same proportions. The most common typing procedurescurrently used with Shigella spp. are plasmid analysis and
PFGE (7, 8, 12, 13, 24). Shigella species usually harbor a het-
erogenous population of plasmids, ranging in number from 2to as many as 10 (9). Plasmid analysis has proven to be a usefultyping technique (7, 8). Moreover, it is inexpensive and quickto perform, but it can be limiting if we take into account thefact that many plasmids are unstable and may be easily gainedand/or lost. PFGE has a high discriminatory power, although itis cumbersome and expensive. However, it has been widelyused for typing Shigella spp. (13, 24). Taking PFGE as a ref-
erence epidemiological tool, strains belonging to the samePFGE group but having different plasmid profiles and differentantibiograms were observed (for instance, subgroups F
4and
F5). Therefore, the mechanisms of resistance are probably
carried in the missing plasmid. The contrary is also true; twostrains belonging to the same PFGE group with the sameplasmid profile showed different antibiograms (for instance,subgroups F
8and F9). This is probably due to an integron or
transposon carrying the resistance gene integrated in the chro-mosome.
Recently, Liu et al. (13) compared plasmid profiles, PFGE,
and enterobacterial repetitive intergenic consensus PCR fortyping 20 clinical isolates of S. sonnei. PCR-based techniques
have the advantages of being quick and easy to perform, and inthis case they proved to be as good at discriminating epidemi-ologically related strains as PFGE. We found something sim-ilar with REP-PCR, another PCR-based technique, in whichthe amplification of the regions between REP sequences givesa good fingerprinting pattern valid for epidemiological typing.As long as the protocol is strictly followed and conditions arekept constant, this technique provides a degree of discrimina-tion equivalent to that of PFGE with the advantages of speed,simplicity, and economy. To our knowledge, this is the firsttime that such a technique has been used in comparison withPFGE and plasmid profiles to type different species of Shigella.
Antimicrobial susceptibility testing showed a high degree of
resistance to antibiotics most commonly used in the area (tet-racycline, ampicillin, co-trimoxazole, and chloramphenicol).No resistance to quinolones and cephalosporins was observed,
FIG. 3. Plasmid patterns. Lane 1, S. flexneri strain belonging to subgroup F2;
lanes 2 and 3, S. flexneri strains belonging to subgroup F1; lane 4, S. flexneri strain
belonging to subgroup F3; lanes 5 and 6, S. flexneri strains belonging to subgroup
F4; lane 7, S. flexneri strain belonging to subgroup F5; lane 8, S. flexneri strain
belonging to subgroup F6; lane 9, S. flexneri strain belonging to subgroup F7;
lanes 10 and 11, S. flexneri strains belonging to subgroups F8and F9; lane 12, S.
dysenteriae; lanes 13 and 14, S. sonnei strains belonging to subgroups S1and S2.TABLE 1. Characterization of Shigella spp. by four different
epidemiological markers
SpeciesGroupa
(no.)Subgroupb
(no.)PFGEAntibio-
gramcPlasmid
profileREP-PCR
group
S. flexneri F-I (51) F1(46) A I b 1
F2(4) A I a 1
F3(1) A II c 1
F-II (17) F4(14) B I d 2
F5(3) B VI e 2
F-III (7) F6(5) C I f 3
F7(2) C III g 3
F-IV (3) F8(1) D IV h 4
F9(2) D III h 4
S. sonnei S-I (4) S1(3) F IV j 5
S2(1) F V j 5
S. dysenteriae D-I (4) D1(4) E I i 6
aDistribution of strains based on PFGE and REP-PCR.
bDistribution of groups according to antibiogram and plasmid analyses.
cSee text for phenotypes.VOL. 37, 1999 MECHANISMS OF RESISTANCE OF SHIGELLA SPP. 3115 on December 3, 2019 by guest http://jcm.asm.org/ Downloaded from

which can be explained by the fact that they are not used as
alternative therapies in this area due to their high cost and lackof availability. However, a trend to quinolone resistance hasbeen observed by Ries et al. (20) in S. dysenteriae strains iso-
lated in Burundi. S. dysenteriae is considered the most resistant
of the Shigella spp. (21). However, in our study S. flexneri
showed the same level of resistance as S. dysenteriae. This
pattern of resistance and susceptibility is commonly seen indeveloping countries, in contrast with strains from developedcountries, which are less resistant to these antimicrobial agents(4, 27). In this study, the antimicrobial resistance pattern is nota useful epidemiological marker, due to the lack of variabilityin susceptibility patterns (i.e., the high level of resistanceshown by most isolates). Resistance to ampicillin in S. flexneri
groups F
1and F2and S. dysenteriae (group D) is explained by
the presence of an OXA-1-type b-lactamase within an inte-
gron. Group F3S. flexneri and the one ampicillin-resistant S.
sonnei (group S2) isolate had a TEM-1-type b-lactamase. Both
genes have been previously described in Shigella strains iso-
lated in Denmark and Greece (14, 22). Therefore, this is themost frequent mechanism of ampicillin resistance found inShigella.
Besides ampicillin, the drug of choice for treating shigellosis
is co-trimoxazole. Eighty-eight percent of the strains studiedshowed resistance to this drug, and in most cases this resistancecould be explained by the presence of a dhfr Ia gene previously
described in Shigella and considered the most common di-
hydrofolate reductase gene in the genus. In one group ofS. flexneri, however, the dhfr gene found was dhfr VII, first
described in E. coli (1). These genes were found inserted in an
integron. Both genes were detected with specific primers toamplify the entire gene, which was further sequenced, showingin both cases 100% homology with the dhfr Ia and dhfr VII
genes previously described (19, 23). Chloramphenicol resis-tance was explained in every case by a positive chloramphen-icol acetyltransferase activity generating a high level of resis-tance. The use of this antibiotic has rapidly declined in manycountries. However, due to the fact that it is inexpensive andpresents a broad-spectrum activity it is extensively employed indeveloping countries, thereby ensuring strong selection pres-sure for the maintenance of chloramphenicol resistance.
In this study, we suggest that antibiotic resistance determi-
nants are carried by plasmids, as well as in integrons whichcontain resistance genes, such as bla
OXA ordhfr genes. The
spread of multiresistant Shigella strains among a population in
which diarrheal disease is one of the major causes of childmorbidity and mortality requires greater attention to the ap-propriate use of antibiotics, the establishment of hygienic mea-sures to prevent or decrease transmission, and the develop-ment of new effective drugs that can be safely used withchildren. Moreover, the guidelines for the treatment of shig-ellosis in developing countries should be updated, since in thisstudy co-trimoxazole, one of the recommended antimicrobialagents for the treatment of shigellosis, has been shown to havelittle activity against Shigella spp.
ACKNOWLEDGMENTS
We thank the parents and guardians of study participants and the
staff of the Ifakara Health Research and Development Centre and the
St. Francis Designated District Hospital. Particular thanks go to theclinical officers whose work was of central importance.
This work was supported in part by grant SAF97/0091 and the
Spanish Agency for International Co-operation (AECI-1042).
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