# Virulence Factors Associated with Pediatric Shigellosis in Brazilian Amazon

Authors: Carolinie Batista Nobre da Cruz; Maria Carolina Scheffer de Souza; Paula Taquita Serra; Ivanildes Santos; Antonio Balieiro; Fabio Alessandro Pieri; Paulo Afonso Nogueira; Patrícia Puccinelli Orlandi
Journal: BioMed Research International
 (2014)

---

## Abstract

Shigellosis is a global human health problem and the incidence is highest among children. In the present work, mainShigella virulence genes was examined by PCR and compared to symptoms of pediatric shigellosis. Thirty Shigella isolates were identified from an etiologic study at which 1,339 children ranging 0–10 years old were enrolled. S. flexneri was the most frequent species reaching 60.0% of isolates, 22.2% were S. sonnei, and 6.6% were both S. dysenteriae and S. boydii. All Shigella infected children had diarrhea, but not all were accompanied by others symptoms of bacillary dysentery. Among major virulence genes, the PCR typing revealed ipaBCD was present in all isolates, followed by IpaH7.8, set-1A, set-1B, sen/ospD3, virF, and invE. The pathogenic potential of the ShET-1B subunit was observed in relation to dehydration (P
<
0.001) and ShET-2 related to the intestinal injury (P
=
0.033) evidenced by the presence of bloody diarrhea. Our results show associations among symptoms of shigellosis and virulence genes of clinical isolates of Shigella spp.


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## Body

## 1. Introduction

Shigella spp. is Gram-negative bacilli of the Enterobacteriaceae family that are perfectly adapted to colonize the host intestine subverting the host’s defenses in their favor [1–4].The genusShigella encompasses four subgroups historically treated as species:Shigella flexneri,Shigella boydii, Shigella sonnei, andShigella dysenteriae [5]. These species are the etiological agents of bacillary dysentery or shigellosis, manifested by fever, small volume of bloody, mucoid stools; abdominal cramps; and mucoid, bloody diarrhea [1, 6]. Other clinical manifestations range between nausea, vomiting, and dehydration. Depending on the virulence potential of the strain and the nutritional status of the individual, shigellosis can progress to severe disease when accompanied by rectal tenesmus, with neurological symptoms such as headache and lethargy [1].Shigella virulence is based on the presence of a large virulenceinv plasmid, carrying an operon that encodes the type III-secretion-system (T3SS) responsible for bacterial entry [7, 8]. Theial gene is found oninv plasmid and invasion-related processes [9]. The T3SS is composed of several proteins, including a needle shape oligomer anchored in the protein complex which connects the inner and outer bacterial membranes. The tip of the needle is oligomer composed for invasion plasmid antigens,ipaB,ipaC, andipaD [6–9]. TheipaH gene is present as multiple copies, five on large plasmid and seven on chromosome. One of five copies, theipaH7.8, plays a role in modulating the inflammatory response elicited by infection and shares a conserved C-terminal novel E3 ligase (C-term-E3-ligase) and variable N-terminal leucine-rich repeat (LRR) domains [10].Others genes are important bacterial pathogenicity factors in the intestinal tract, such as the enterotoxins that have significant enterotoxic activityin vitro when tested in rabbit ileal loops and Ussing chambers [1].Shigella strains produce distinct enterotoxins:Shigella enterotoxin 1 (ShET-1) chromosome encoded byset1A which is present in allS. flexneri 2a.Shigella enterotoxin 2 (ShET-2) encoded by genesen/ospD3located on a large plasmid associated with virulence ofShigella and found in many, but not all,Shigella of different serotypes and also in enteroinvasiveEscherichia coli (EIEC) [9, 11]. And two distinct Shiga toxins (Stx-1 and Stx-2) are encoded by chromosomal genes and expressed byS. dysenteriae and similar to the Shiga-like toxins of enterohemorrhagicE. coli [1].The mechanisms of main pathogenic factors ofShigellaare well stablished; however, studies focusing association between pathogenicity factors and shigellosis symptoms in human are scarce [12, 13]. In this work, the major virulence genes ofShigella species derived from pediatric bacillary dysentery were examined for PCR and the goal of this study was to investigate the relationship with symptoms of shigellosis.


## 2. Material and Methods

Patients and Samples. During a period from August 2007 to December 2008, stool specimens were collected from 1339 children ranging 0–10 years old who sought treatment at three hospitals, in Manaus, in the center of Brazilian Amazon, and transferred to a clinical microbiology laboratory. An axillary temperature higher than 37.8°C was considered fever when determined at the time of clinical assessment or as reported by the child’s guardian. Dehydration was diagnosed by the attending medical professional. The presence or absence of vomiting was reported by the individual responsible for the clinical evaluation. The child’s guardian was first informed about the research and asked to participate by filling out a consent form and a case report form (Ethics Committee of the Federal University of Amazonas 266/206). The inclusion criteria were as follows: the age of the patients was in the range of 0–10 years old, the patients had diarrhea that lasted 7 days, and blood was evident by stool examination with a fecal occult blood (FOB) test using the Feca-Cult Kit (Inlab diagnostica). The present study was designed to isolateShigella strains from clinical samples of patients with bloody diarrhea by culture methods and characterize them by appropriate biochemical and serological tests.Bacterial Culture, Isolation, and Antibiogram. Lactose nonfermenting colonies were selected on MacConkey lactose agar (MC),Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) agar, and Shigella species were identified by biochemical panel that consisted of EPM and MiLi-citrate. A total of 36 isolates ofShigellaspp. were identified. TheShigella flexneri M90T was used as reference strains for comparison purposes. The antibiogram technique was performed as described by [14]. The following antibiotics were tested: amikacin (AMK), amoxicillin + clavulanic acid (AMC), ampicillin (AMP), ciprofloxacin (CIP), chloramphenicol (CLO), ceftriaxone (CRO), gentamicin (GEN), kanamycin (K), nalidixic acid (NAL), and tetracycline (TET).Serological Tests. TheShigella strains were subcultured on MacConkey agar plates, and serological tests were performed by the slide agglutination method. The serotypes of allShigella isolates were determined with commercially variable polyclonal antisera (Promicro-Brazil) against allShigella serotypes, includingS. sonnei 1 and 2, polyvalentS. flexneri,S. dysenteriae 2, andS. boydii 11.PCR Assays. Each sample was submitted to PCR amplification with ten pairs of different primers (Table 1). For the detection of virulence genes, DNA was extracted from the samples using the phenol-chloroform method. Ten pairs of primers corresponding to the genusShigella and two primers (uidA andinvE) corresponding to invasion genes that are also found inEscherichia coli were used. The primers sequences used were obtained from Invitrogen, Brazil. Descriptions and the sequences of the PCR primers used in this study are given in Table 1. The primers foripaH7.8 annealed a specific region that overlapped two contiguous genes, LRR and C-term-E3-ligase genes. The primers foripaBCD amplified a product from lociIpa located upstream toipaB gene. Amplification was performed in a thermocycler (Eppendorf, Germany) by the methods described by Aranda et al. [13] and Faruque et al. [15]. The expected sizes of the amplicons were ascertained by electrophoresis in 1.5% agarose gel with an appropriate molecular size marker (Promega, Brazil).Table 1
The striking points employed for the detection of virulence markers ofShigella.
Gene
Amplicon size (bp)
Primer
Annealing temperature °C
Reference
evt
100
CAACACTGGATGATCTCAG
56
[15]
CCCCCTCAACTGCTAATA
ial
320
CTGGATGGTATGGTGAGG
60
[18]
GGAGGCCAACAATTATTTCC
ipaBCD
500
GCTATAGCAGTGACATG
59
[15]
ACGAGTTCGAAGCACTC
ipaH
933
CTCGGCACGTTTTAATAGTCTGG
59
[19]
GTGGAGAGCTGAAGTTTCTCTGC
set1A
309
TCACGCTACCATCAAAGA
57
[18]
TATCCCCCTTTGGTGGTA
set1B
147
GTGAACCTGCTGCCGATATC
57
[18]
ATTAGTGGATAAAAATGACG
sen/ospD3
799
ATGTGCCTGCTATTATTTAT
52
[18]
CATAATAATAAGCGGTCAGC
virF
618
TCAGGCAATGAAACTTTGAC
60
[19]
TGGGCTTGATATTCCGATAAGTC
uidA
1487
ATGCCAGTCCAGCGTTTTTGC
54
[20]
AAAGTGTGGGTCAATAATCAGGAAGTG
invE
766
CGATAGATGGCGAGAAATTATATCCCG
56
[20]
CGATCAAGAATCCCTAACAGAAGAATCACThe reactions were performed under the following conditions: 40 ng of DNA, 5X buffer, 0.25 mM dNTPs, 2.5 mM MgCl2, 5 μM of each primer, 2.5 U of high-fidelity Taq DNA polymerase (Invitrogen), and sterile deionized water in a total volume of 12.5 μL. PCR was performed in a thermocycler (Eppendorf) and consisted of the following steps: 94°C for 3 minutes, followed by 30 cycles of 94°C for 30 seconds, varying annealing temperatures for each gene (Table 1) for 45 seconds, and 72°C for 1 minute and 30 seconds. The final extension step was performed at 72°C for 10 minutes, followed by cooling to 4°C. The fragments obtained were analyzed by horizontal electrophoresis on a 1% agarose gel at 100 V in TBE buffer. The gel was stained in a solution of ethidium bromide and visualized on a transilluminator.16S rRNA Gene Sequencing. To confirmShigella species identification, a region from 16S rRNA gene located between 530° to 1492° nucleotides was amplified using the primers forward 5′-TGA CTG ACT GAG TGC CAG CMG CCG CGG-3′ and reverse 5′-TGA CTG ACT GAG AGC TCT ACC TTG TTA CGM YTT-3′ [16, 17]. The reaction (50 mM MgSO4, 0.5 μL of 10 mM dNTPs, 5 pmol of each primer, 1.25 U Platinum Taq DNA polymerase High Fidelity, 10x buffer) consisted of three cycles (1x 94°C for 2 min; 35x 94°C for 30 s; 58°C for 30 s; and 1x 68°C for 1 min). After edition, the taxonomic affiliation was performed with “Ribosomal Database Project II” database. A minimum of 75% similarity was considered for the encountered species.


## 3. Results

### 3.1. Diarrhea Symptoms Related toShigella Infections

In the present study, thirtyShigella species were isolated from an etiologic study at which 1,339 children presenting with diarrhea over the period from August 2007 to July 2008.Shigella species were the fifth most common cause of diarrhea (2.2%), that were led by enteropathogenicEscherichia coli in 837 cases (62.1%), followed by 207 children withRotavirus (15.4%) and 192 withSalmonella species (14.3%), and 34 casesof Yersinia species (2.5%). Protozoa infection was observed in 46 cases:Entamoeba histolyticawas found in 16 cases, 14 forGiardia lamblia, 13 forEntamoeba coli, and 3 forBalantidium coli. Twenty-four children had diarrhea associated with worms, 9 forEnterobius vermiculares, 9 forAscaris lumbricoides, 4 forAncylostoma species, and 2 forTrichiura trichuris. And still, the diarrhea etiology of one hundred ninety-nine children was unknown.Monoinfections among major groups of enteropathogens were found, bacteria (N
=
867), rotavirus (N
=
39), and intestinal parasites (N
=
8). Several coinfections were also found; thirteen children were infected by enteropathogenic bacteria, rotavirus, and intestinal parasites. Enteropathogenic bacteria coinfected with rotavirus in one hundred sixty-eight cases or with intestinal parasites in forty-five children were found.Although rainfall in the region is seasonal [21], the temporal variation of cases ofShigella diarrhea did not fluctuate during the two rainfall stations, unlike the cases of diarrhea by other enteropathogens, which increased over the rainy season (Figure 1).Figure 1
Temporal variation in diarrhea prevalence caused byShigella and others enteropathogens. From August 2007 to December 2008, 1346 children in the range of 0–10 years old were admitted to hospital with diarrhea and they sought treatment at three hospitals in Manaus, in central of Brazilian Amazon. Stool specimens were collected at whichShigella as much as other enteropathogens were identified by classical methods. Distribution of rainfall over period of study is classified in two levels. Dark gray rectangles were the highest rate of rainfall (between 300 and 600 mm). Light gray indicates the rainfall that was below 200 mm [21].Thestudy was carried out with children aged 0–10 years and as expected children over 2 years of age were moreaffected byShigella (P
=
0.002). The median of age of children affected byShigella was 24 months (ranging from 14.2 to 47.2) differing from the group affected by other enteropathogens (14 months, ranging from 8 and 25). With respect to other epidemiologic factors, no difference was observed in both groups regarding the number and duration of diarrhea as well as the quality of the water consumed by population.To characterize the main symptoms related toShigella infections, initially the main diarrhea symptoms were compared among most prevalent etiologies (Table 2). The frequency of febrile children and dehydration signs were high and independent of etiology as expected. Similarly, the frequencies of children who have reported vomiting in clinical assessment were also high, except bacteria and rotavirus coinfected children whose frequency was slight higher (P
=
0.006). In contrast, low frequencies of blood in stool and fecal occult blood were found among children independent of etiology, with even lower frequencies among coinfected children by rotavirus and bacteria or rotavirus monoinfection children (P
=
0.009).Table 2
Comparison of diarrhea symptoms among etiologic agents.
Symptoms
Bacteria monoinfection
Parasite and bacterial Coinfection
RV and bacterial coinfection
RV monoinfection
Unknown etiology
P
No bacteria as etiologic agent
E. coli
Salmonella
Shigella
Yersínia
P
N
=
867
N
=
45
N
=
168
N
=
39
N
=
199
N
=
246
N
=
837
N
=
192
N
=
30
N
=
34
Fever
Pos.
646 (74.5)
31 (68.9)
127 (75.6)
34 (87.2)
148 (74.4)
0.185
189 (76.8)
615 (73.5)
144 (75)
25 (83.3)
28 (82.4)
0.588
Neg.
218 (25.1)
14 (31.1)
40 (23.8)
4 (10.3)
51 (25.6)
56 (22.8)
220 (26.3)
46 (24)
5 (16.7)
6 (17.6)
NI#
3 (0.3)
0 (0)
1 (0.6)
1 (2.6)
0 (0)
1 (0.4)
2 (0.2)
2 (1)
0 (0)
0 (0)
Vomiting
Pos.
633 (73)
31 (68.9)
143 (85.1)
33 (84.6)
148 (74.4)
0.006
186 (75.6)
640 (76.5)
136 (70.8)
16 (53.3)
26 (76.5)
0.036
Neg.
231 (26.6)
14 (31.1)
24 (14.3)
5 (12.8)
51 (25.6)
59 (24)
195 (23.3)
55 (28.6)
13 (43.3)
8 (23.5)
NI
3 (0.3)
0 (0)
1 (0.6)
1 (2.6)
0 (0)
1 (0.4)
2 (0.2)
1 (0.5)
1 (3.3)
0 (0)
Dehydration
Pos.
590 (68.1)
26 (57.8)
104 (61.9)
28 (71.8)
137 (68.8)
0.07
167 (67.9)
559 (66.8)
130 (67.7)
20 (66.7)
21 (61.8)
0.832
Neg.
239 (27.6)
19 (42.2)
54 (32.1)
9 (23.1)
60 (30.2)
72 (29.3)
238 (28.4)
55 (28.6)
10 (33.3)
12 (35.3)
NI
38 (4.4)
0 (0)
10 (6)
2 (5.1)
2 (1)
7 (2.8)
40 (4.8)
7 (3.6)
0 (0)
1 (2.9)
Blood in stool
Pos.
134 (15.5)
3 (6.7)
20 (11.9)
3 (7.7)
38 (19.1)
0.312
46 (18.7)
118 (14.1)
24 (12.5)
10 (33.3)
5 (14.7)
0.074
Neg.
718 (82.8)
41 (91.1)
145 (86.3)
36 (92.3)
159 (79.9)
198 (80.5)
704 (84.1)
165 (85.9)
19 (63.3)
29 (85.3)
NI
15 (1.7)
1 (2.2)
3 (1.8)
0 (0)
2 (1)
2 (0.8)
15 (1.8)
3 (1.6)
1 (3.3)
0 (0)
Fecal occult blood
Pos.
227 (26.2)
13 (28.9)
25 (14.9)
5 (12.8)
53 (26.6)
0.009
61 (24.8)
208 (24.9)
34 (17.7)
18 (60)
8 (23.5)
<0.001
Neg.
640 (73.8)
32 (71.1)
143 (85.1)
34 (87.2)
146 (73.4)
185 (75.2)
629 (75.1)
158 (82.3)
12 (40)
26 (76.5)
Frequencies were calculated by the Chi-square test.
#NI: not informed.Regarding four enterobacteria, independently the analyses were performed with same symptoms. The frequency of febrile children and dehydration signs were high and independent of bacteria species or others etiologic agents. Also in relation to blood in stool, low frequencies and none difference were found. Differences were found regarding vomiting and fecal occult blood. AmongShigella infected children, the frequency of those who have reported vomiting in clinical assessment was lower in relation to others bacteria (P
=
0.036) including coinfection groups (Table 2).The main difference concerned fecal occult blood, while with all etiologic agents the presence of traces of blood in stool had been less frequent, and the number ofShigella infected children was higher than expected (P
<
0.001). Thus, only with one accurate method traces of blood in stool might associate with bacillary dysentery (Table 2).


### 3.2. Virulence Genes Related to Pediatric Shigellosis

The conventional and 16S ribosomal gene confirmed 18 isolates ofS. flexneri (8S. sonnei, 2S. dysenteriae,and 2S. boydii isolates). The antimicrobial resistance was 80.0% (24/30) to tetracycline, 40.0% (12/30) to ampicillin, 30.0% (9/30) to chloramphenicol, 30.0% (9/30) to gentamicin, and 13.0% (4/30) to both antibiotics amikacin and clavulanic acid. Thus, the resistance to ciprofloxacin and ceftriaxone was lower, with only 3% (1/30) of isolates presenting resistance. All isolates were sensitive to kanamycin and nalidixic acid (Table 3).Table 3
Frequencies and distribution of virulence genes and antimicrobial resistance ofShigella spp. and symptoms presented by children.
Isolates
Shigella species by 16S RNA gene
ipaBCD
ipaH
set-1A
set-1B
Sen/ospD3
ial
virF
evt
invE
Antimicrobial resistance
Vomiting
Dehydration
Blood in stool
Fecal Occult Blood
2
flexneri
+
+
+
+
+
53
dysenteriae
+
+
tet
80
flexneri
+
+
+
+
+
amp, amk, amc, clo, tet
+
+
85
flexneri
+
+
+
+
+
+
+
amp, clo, tet
+
+
+
97
flexneri
+
+
+
+
+
+
amp, clo, tet
+
113
flexneri
+
+
+
clo, tet
+
+
183
sonnei
+
+
+
+
gen, tet
+
190
dysenteriae
+
+
+
+
192
boydii
+
+
+
+
+
+
tet
199
flexneri
+
+
+
+
+
+
tet
+
+
201
flexneri
+
+
+
+
+
+
+
amp, cef, tet
+
202
flexneri
+
+
+
amp, tet
+
279
sonnei
+
+
+
+
tet
+
+
337
flexneri
+
+
+
+
+
+
+
539
sonnei
+
+
+
tet
+
+
562
sonnei
+
+
+
+
+
+
+
gen, tet
+
+
+
586
sonnei
+
+
+
+
+
+
+
amp, amk, amc, clo, tet
+
+
625
flexneri
+
+
+
+
+
+
+
amp, cip, clo, tet
+
+
+
837
flexneri
+
+
+
+
tet
+
+
+
+
873
flexneri
+
+
+
+
tet
+
+
+
883
sonnei
+
+
+
+
amp, tet
+
+
+
893
flexneri
+
+
+
+
+
gen, tet
+
956
boydii
+
+
+
+
+
+
+
tet
+
+
+
1039
flexneri
+
+
+
amp, clo, tet
+
+
+
+
1065
flexneri
+
+
amp, clo,
+
+
1118
flexneri
+
+
+
+
+
+
+
+
tet
+
+
+
+
1124
sonnei
+
+
+
amp, tet
+
+
+
+
1163
flexneri
+
+
+
+
1234
sonnei
+
+
+
+
+
+
+
+
amp, clo, tet
+
+
+
1257
flexneri
+
+
+
+
+
+
gen
+
Frequencies
100.0
63.3
63.3
56.7
43.3
56.7
43.3
10.0
43.3
53.3
66.7
33.3
60.0
+: Positive.
Abbreviations of antibiotics tested: amk: amikacin, amc: amoxicillin/clavulanic acid, amp: ampicillin, cip: ciprofloxacin, clo: chloramphenicol, cro: ceftriaxone,  gen: gentamicin,  and tet: tetracycline.The detection of some majorShigella virulence genes gave intense amplicons with a clean background in each reaction according to conditions and PCR products (Table 1). TheipaBCD gene was present in all isolates. Concerning others virulence genes, a vast genetic diversity was shown among isolates;ipaH andset-1A genes were predominant in 63.3% of the isolates (19/30), followed byset-1B andial in 56.7% (17/30) of the isolates (Table 3). Thesen/ospD3 (ShET-2),virF, andinvE genes were present at a frequency of 43.3%, that is, in 13 isolates. Still, theevt was detected in 3 isolates (10.0%), despite the low frequency ofS. dysenteriae. The presence ofevt gene and antimicrobial resistance of the isolates are shown together with the symptoms presented by children (Table 3). Some isolates carriedset-1A but notset-1B, or vice versa.The high frequencies ofipaBCD andipaH genes could explain frequencies of fever, vomiting, and dehydration in infected children. Regardless ofvirF, invE, andevt genes due low frequencies, the analyses were performed withialand (invasion-related processes) andset1-A andset-1B. No association was found with fever, vomiting, or blood in stool with genes (data not shown).In contrast, presence of blood traces in stool was related to shigellosis, and less common to all etiologic agents, two associations concerningShigellaenterotoxins were found. TheShigella species carryingsen/ospD3 gene for ShET-2 enterotoxin hemolysin were more frequent in children that had traces of blood in stools (P
=
0.042). And a strong association was found with dehydration andset1-B gene forShigella enterotoxin 1 (P
<
0.001) known for causing watery phase of diarrhea (Table 4). Thus, the PCR typing permitted us to connect particular virulence genes with symptoms of pediatric shigellosis.Table 4
Assessing of majorShigella virulence genes associated with main symptoms of dysentery bacillary.
Virulence gene
Dehydration
Prevalence ratio
CI
P
Fecal occult blood
Prevalence  ratio
CI
P
Pos.
Neg.
Pos.
Neg.
ial
Pos.
12 (60)
5 (50)
1.15
(0.68–1.94)
0.705
11 (61.1)
6 (50)
1.2
(0.65–2.22)
0.821
Neg.
8 (40)
5 (50)
7 (38.9)
6 (50)
ipaH
Pos.
16 (80)
8 (80)
1
(0.53–1.88)
0.999
16 (88.9)
8 (66.7)
2
(0.62–6.42)
0.184
Neg.
4 (20)
2 (20)
2 (11.1)
4 (33.3)
set.1A
Pos.
14 (70)
5 (50)
1.35
(0.74–2.47)
0.425
12 (66.7)
7 (58.3)
1.16
(0.61–2.19)
0.712
Neg.
6 (30)
5 (50)
6 (33.3)
5 (41.7)
set.1B
Pos.
16 (80)
1 (10)
3.06
(1.34–6.97)
<0.001
**
10 (55.6)
7 (58.3)
0.96
(0.53–1.72)
0.999
Neg.
4 (20)
9 (90)
8 (44.4)
5 (41.7)
sen/ospD3
Pos.
9 (45)
4 (40)
1.07
(0.65–0.77)
0.999
11 (61.1)
2 (16.7)
2.05
(1.11–3.80)
0.042
*
Neg.
11 (55)
6 (60)
7 (38.9)
10 (83.3)
P value of Fisher's exact test. **significant difference.


## 3.1. Diarrhea Symptoms Related toShigella Infections

In the present study, thirtyShigella species were isolated from an etiologic study at which 1,339 children presenting with diarrhea over the period from August 2007 to July 2008.Shigella species were the fifth most common cause of diarrhea (2.2%), that were led by enteropathogenicEscherichia coli in 837 cases (62.1%), followed by 207 children withRotavirus (15.4%) and 192 withSalmonella species (14.3%), and 34 casesof Yersinia species (2.5%). Protozoa infection was observed in 46 cases:Entamoeba histolyticawas found in 16 cases, 14 forGiardia lamblia, 13 forEntamoeba coli, and 3 forBalantidium coli. Twenty-four children had diarrhea associated with worms, 9 forEnterobius vermiculares, 9 forAscaris lumbricoides, 4 forAncylostoma species, and 2 forTrichiura trichuris. And still, the diarrhea etiology of one hundred ninety-nine children was unknown.Monoinfections among major groups of enteropathogens were found, bacteria (N
=
867), rotavirus (N
=
39), and intestinal parasites (N
=
8). Several coinfections were also found; thirteen children were infected by enteropathogenic bacteria, rotavirus, and intestinal parasites. Enteropathogenic bacteria coinfected with rotavirus in one hundred sixty-eight cases or with intestinal parasites in forty-five children were found.Although rainfall in the region is seasonal [21], the temporal variation of cases ofShigella diarrhea did not fluctuate during the two rainfall stations, unlike the cases of diarrhea by other enteropathogens, which increased over the rainy season (Figure 1).Figure 1
Temporal variation in diarrhea prevalence caused byShigella and others enteropathogens. From August 2007 to December 2008, 1346 children in the range of 0–10 years old were admitted to hospital with diarrhea and they sought treatment at three hospitals in Manaus, in central of Brazilian Amazon. Stool specimens were collected at whichShigella as much as other enteropathogens were identified by classical methods. Distribution of rainfall over period of study is classified in two levels. Dark gray rectangles were the highest rate of rainfall (between 300 and 600 mm). Light gray indicates the rainfall that was below 200 mm [21].Thestudy was carried out with children aged 0–10 years and as expected children over 2 years of age were moreaffected byShigella (P
=
0.002). The median of age of children affected byShigella was 24 months (ranging from 14.2 to 47.2) differing from the group affected by other enteropathogens (14 months, ranging from 8 and 25). With respect to other epidemiologic factors, no difference was observed in both groups regarding the number and duration of diarrhea as well as the quality of the water consumed by population.To characterize the main symptoms related toShigella infections, initially the main diarrhea symptoms were compared among most prevalent etiologies (Table 2). The frequency of febrile children and dehydration signs were high and independent of etiology as expected. Similarly, the frequencies of children who have reported vomiting in clinical assessment were also high, except bacteria and rotavirus coinfected children whose frequency was slight higher (P
=
0.006). In contrast, low frequencies of blood in stool and fecal occult blood were found among children independent of etiology, with even lower frequencies among coinfected children by rotavirus and bacteria or rotavirus monoinfection children (P
=
0.009).Table 2
Comparison of diarrhea symptoms among etiologic agents.
Symptoms
Bacteria monoinfection
Parasite and bacterial Coinfection
RV and bacterial coinfection
RV monoinfection
Unknown etiology
P
No bacteria as etiologic agent
E. coli
Salmonella
Shigella
Yersínia
P
N
=
867
N
=
45
N
=
168
N
=
39
N
=
199
N
=
246
N
=
837
N
=
192
N
=
30
N
=
34
Fever
Pos.
646 (74.5)
31 (68.9)
127 (75.6)
34 (87.2)
148 (74.4)
0.185
189 (76.8)
615 (73.5)
144 (75)
25 (83.3)
28 (82.4)
0.588
Neg.
218 (25.1)
14 (31.1)
40 (23.8)
4 (10.3)
51 (25.6)
56 (22.8)
220 (26.3)
46 (24)
5 (16.7)
6 (17.6)
NI#
3 (0.3)
0 (0)
1 (0.6)
1 (2.6)
0 (0)
1 (0.4)
2 (0.2)
2 (1)
0 (0)
0 (0)
Vomiting
Pos.
633 (73)
31 (68.9)
143 (85.1)
33 (84.6)
148 (74.4)
0.006
186 (75.6)
640 (76.5)
136 (70.8)
16 (53.3)
26 (76.5)
0.036
Neg.
231 (26.6)
14 (31.1)
24 (14.3)
5 (12.8)
51 (25.6)
59 (24)
195 (23.3)
55 (28.6)
13 (43.3)
8 (23.5)
NI
3 (0.3)
0 (0)
1 (0.6)
1 (2.6)
0 (0)
1 (0.4)
2 (0.2)
1 (0.5)
1 (3.3)
0 (0)
Dehydration
Pos.
590 (68.1)
26 (57.8)
104 (61.9)
28 (71.8)
137 (68.8)
0.07
167 (67.9)
559 (66.8)
130 (67.7)
20 (66.7)
21 (61.8)
0.832
Neg.
239 (27.6)
19 (42.2)
54 (32.1)
9 (23.1)
60 (30.2)
72 (29.3)
238 (28.4)
55 (28.6)
10 (33.3)
12 (35.3)
NI
38 (4.4)
0 (0)
10 (6)
2 (5.1)
2 (1)
7 (2.8)
40 (4.8)
7 (3.6)
0 (0)
1 (2.9)
Blood in stool
Pos.
134 (15.5)
3 (6.7)
20 (11.9)
3 (7.7)
38 (19.1)
0.312
46 (18.7)
118 (14.1)
24 (12.5)
10 (33.3)
5 (14.7)
0.074
Neg.
718 (82.8)
41 (91.1)
145 (86.3)
36 (92.3)
159 (79.9)
198 (80.5)
704 (84.1)
165 (85.9)
19 (63.3)
29 (85.3)
NI
15 (1.7)
1 (2.2)
3 (1.8)
0 (0)
2 (1)
2 (0.8)
15 (1.8)
3 (1.6)
1 (3.3)
0 (0)
Fecal occult blood
Pos.
227 (26.2)
13 (28.9)
25 (14.9)
5 (12.8)
53 (26.6)
0.009
61 (24.8)
208 (24.9)
34 (17.7)
18 (60)
8 (23.5)
<0.001
Neg.
640 (73.8)
32 (71.1)
143 (85.1)
34 (87.2)
146 (73.4)
185 (75.2)
629 (75.1)
158 (82.3)
12 (40)
26 (76.5)
Frequencies were calculated by the Chi-square test.
#NI: not informed.Regarding four enterobacteria, independently the analyses were performed with same symptoms. The frequency of febrile children and dehydration signs were high and independent of bacteria species or others etiologic agents. Also in relation to blood in stool, low frequencies and none difference were found. Differences were found regarding vomiting and fecal occult blood. AmongShigella infected children, the frequency of those who have reported vomiting in clinical assessment was lower in relation to others bacteria (P
=
0.036) including coinfection groups (Table 2).The main difference concerned fecal occult blood, while with all etiologic agents the presence of traces of blood in stool had been less frequent, and the number ofShigella infected children was higher than expected (P
<
0.001). Thus, only with one accurate method traces of blood in stool might associate with bacillary dysentery (Table 2).


## 3.2. Virulence Genes Related to Pediatric Shigellosis

The conventional and 16S ribosomal gene confirmed 18 isolates ofS. flexneri (8S. sonnei, 2S. dysenteriae,and 2S. boydii isolates). The antimicrobial resistance was 80.0% (24/30) to tetracycline, 40.0% (12/30) to ampicillin, 30.0% (9/30) to chloramphenicol, 30.0% (9/30) to gentamicin, and 13.0% (4/30) to both antibiotics amikacin and clavulanic acid. Thus, the resistance to ciprofloxacin and ceftriaxone was lower, with only 3% (1/30) of isolates presenting resistance. All isolates were sensitive to kanamycin and nalidixic acid (Table 3).Table 3
Frequencies and distribution of virulence genes and antimicrobial resistance ofShigella spp. and symptoms presented by children.
Isolates
Shigella species by 16S RNA gene
ipaBCD
ipaH
set-1A
set-1B
Sen/ospD3
ial
virF
evt
invE
Antimicrobial resistance
Vomiting
Dehydration
Blood in stool
Fecal Occult Blood
2
flexneri
+
+
+
+
+
53
dysenteriae
+
+
tet
80
flexneri
+
+
+
+
+
amp, amk, amc, clo, tet
+
+
85
flexneri
+
+
+
+
+
+
+
amp, clo, tet
+
+
+
97
flexneri
+
+
+
+
+
+
amp, clo, tet
+
113
flexneri
+
+
+
clo, tet
+
+
183
sonnei
+
+
+
+
gen, tet
+
190
dysenteriae
+
+
+
+
192
boydii
+
+
+
+
+
+
tet
199
flexneri
+
+
+
+
+
+
tet
+
+
201
flexneri
+
+
+
+
+
+
+
amp, cef, tet
+
202
flexneri
+
+
+
amp, tet
+
279
sonnei
+
+
+
+
tet
+
+
337
flexneri
+
+
+
+
+
+
+
539
sonnei
+
+
+
tet
+
+
562
sonnei
+
+
+
+
+
+
+
gen, tet
+
+
+
586
sonnei
+
+
+
+
+
+
+
amp, amk, amc, clo, tet
+
+
625
flexneri
+
+
+
+
+
+
+
amp, cip, clo, tet
+
+
+
837
flexneri
+
+
+
+
tet
+
+
+
+
873
flexneri
+
+
+
+
tet
+
+
+
883
sonnei
+
+
+
+
amp, tet
+
+
+
893
flexneri
+
+
+
+
+
gen, tet
+
956
boydii
+
+
+
+
+
+
+
tet
+
+
+
1039
flexneri
+
+
+
amp, clo, tet
+
+
+
+
1065
flexneri
+
+
amp, clo,
+
+
1118
flexneri
+
+
+
+
+
+
+
+
tet
+
+
+
+
1124
sonnei
+
+
+
amp, tet
+
+
+
+
1163
flexneri
+
+
+
+
1234
sonnei
+
+
+
+
+
+
+
+
amp, clo, tet
+
+
+
1257
flexneri
+
+
+
+
+
+
gen
+
Frequencies
100.0
63.3
63.3
56.7
43.3
56.7
43.3
10.0
43.3
53.3
66.7
33.3
60.0
+: Positive.
Abbreviations of antibiotics tested: amk: amikacin, amc: amoxicillin/clavulanic acid, amp: ampicillin, cip: ciprofloxacin, clo: chloramphenicol, cro: ceftriaxone,  gen: gentamicin,  and tet: tetracycline.The detection of some majorShigella virulence genes gave intense amplicons with a clean background in each reaction according to conditions and PCR products (Table 1). TheipaBCD gene was present in all isolates. Concerning others virulence genes, a vast genetic diversity was shown among isolates;ipaH andset-1A genes were predominant in 63.3% of the isolates (19/30), followed byset-1B andial in 56.7% (17/30) of the isolates (Table 3). Thesen/ospD3 (ShET-2),virF, andinvE genes were present at a frequency of 43.3%, that is, in 13 isolates. Still, theevt was detected in 3 isolates (10.0%), despite the low frequency ofS. dysenteriae. The presence ofevt gene and antimicrobial resistance of the isolates are shown together with the symptoms presented by children (Table 3). Some isolates carriedset-1A but notset-1B, or vice versa.The high frequencies ofipaBCD andipaH genes could explain frequencies of fever, vomiting, and dehydration in infected children. Regardless ofvirF, invE, andevt genes due low frequencies, the analyses were performed withialand (invasion-related processes) andset1-A andset-1B. No association was found with fever, vomiting, or blood in stool with genes (data not shown).In contrast, presence of blood traces in stool was related to shigellosis, and less common to all etiologic agents, two associations concerningShigellaenterotoxins were found. TheShigella species carryingsen/ospD3 gene for ShET-2 enterotoxin hemolysin were more frequent in children that had traces of blood in stools (P
=
0.042). And a strong association was found with dehydration andset1-B gene forShigella enterotoxin 1 (P
<
0.001) known for causing watery phase of diarrhea (Table 4). Thus, the PCR typing permitted us to connect particular virulence genes with symptoms of pediatric shigellosis.Table 4
Assessing of majorShigella virulence genes associated with main symptoms of dysentery bacillary.
Virulence gene
Dehydration
Prevalence ratio
CI
P
Fecal occult blood
Prevalence  ratio
CI
P
Pos.
Neg.
Pos.
Neg.
ial
Pos.
12 (60)
5 (50)
1.15
(0.68–1.94)
0.705
11 (61.1)
6 (50)
1.2
(0.65–2.22)
0.821
Neg.
8 (40)
5 (50)
7 (38.9)
6 (50)
ipaH
Pos.
16 (80)
8 (80)
1
(0.53–1.88)
0.999
16 (88.9)
8 (66.7)
2
(0.62–6.42)
0.184
Neg.
4 (20)
2 (20)
2 (11.1)
4 (33.3)
set.1A
Pos.
14 (70)
5 (50)
1.35
(0.74–2.47)
0.425
12 (66.7)
7 (58.3)
1.16
(0.61–2.19)
0.712
Neg.
6 (30)
5 (50)
6 (33.3)
5 (41.7)
set.1B
Pos.
16 (80)
1 (10)
3.06
(1.34–6.97)
<0.001
**
10 (55.6)
7 (58.3)
0.96
(0.53–1.72)
0.999
Neg.
4 (20)
9 (90)
8 (44.4)
5 (41.7)
sen/ospD3
Pos.
9 (45)
4 (40)
1.07
(0.65–0.77)
0.999
11 (61.1)
2 (16.7)
2.05
(1.11–3.80)
0.042
*
Neg.
11 (55)
6 (60)
7 (38.9)
10 (83.3)
P value of Fisher's exact test. **significant difference.


## 4. Discussion

From a study in which the etiology of childhood diarrhea was investigated in 1,339 children from periphery of Manaus between August 2007 and July 2009, an intense and heterogeneous amount of enteropathogens found, from monoinfections to coinfections, were found in children from Manaus presenting with diarrhea. The lack of sanitation is a well-known problem in this city because less than 7% of the population has basic sanitation. Shigellosis is a disease that is one of the characteristics of areas like this, where it is difficult to maintain proper hygiene [1, 5, 12, 14, 15, 22–27]; thus, unsurprisingly the indicators of overall mortality and hospital morbidity due to diarrhea in Brazilian children are still worring [26].What is interesting about findings on diarrhea-related symptoms is that independently if diarrhea was caused by mono- or coinfections, frequencies of febrile children, dehydration signs, and vomiting reported in clinical assessment were higher in all enteropathogens groups, and on the other hand frequencies of blood in stool among children were lower (Table2). Moreover, detection of traces of blood in stool was in particular amongShigella-infected children. It is established that infection withShigella can lead to the syndrome of bloody or watery diarrhea; nonetheless, studies, when the information of bloody diarrhea is reported by patients the frequencies, are divergent [28, 29]. Therefore, in the present study, the presence of blood in stool by more accurate method could be evidenced as a particularShigellosis.Shigellosis is an acute intestinal infection, the symptoms of which can range from mild watery diarrhea to severe inflammatory bacillary dysentery [3]. The thirty isolates ofShigella species were confirmed by conventional and 16S rRNA sequencing methods. Our data were consistent with observations in other regions of Brazil, with a predominance ofS.flexneri, followed byS. sonneiorS. boydii, and finallyS. dysenteriae[12, 15, 22–27, 30, 31].Here, some isolates showed resistance to ciprofloxacin and ceftriaxone, which are the antibiotics recommended by the WHO for shigellosis. In contrast, in others studies conducted in North and Northeast of Brazil, allShigella were susceptible to ciprofloxacin and ceftriaxone [24–27]. The emergence of resistantShigella strains might be explained by the indiscriminate use of antimicrobial drugs or treatment failure. Even so, these data contribute to the monitoring of regional strains to ensure the effective treatment of patients and monitoring of the emergence of new resistant strains [24].Despite the fact thatShigellaspecies are considered as the important cause of diarrheal disease, little is known about their genetic diversity worldwide. According to virulence genes examined, theShigellaisolates in this study had a vast genetic diversity. Among mainShigella virulence factors, the T3SS is essential for host cell invasion and intracellular survival [32–34]. The presence ofIpaB,IpaC, andIpaD translocators could be detected using the upstreamipaB region as marker. Our data revealed all the isolates were positive for theipaBCD gene, as expected, whereasIpaB,IpaC, andIpaD are key factors of virulentShigella [3]. UnlikeipaBCD,ipaH 7.8was not very frequent. BecauseipaH 7.8 is present on a large plasmid, this gene would be less stable to storage/subculturing than chromosomal genes encoded byipaH. [35]. Similarly,ipaHwas detected in almost all Shigella species from western Brazilian Amazon [25].Contingency analysis revealedShigella carryingsen/ospD3 was associated to fecal occult blood (P
=
0.042). ShET-2 is known as an enterotoxin hemolysin that elicits inflammatory response duringShigella invasion. Our findings show that in cases ofShigellainfection, ShET-2 contribute to induce intestinal injury induced by inflammation which would lead to bloody diarrhea [3, 8, 9, 11, 36, 37].Regarding ShET-1 enterotoxin, contingency analysis showedShigellaisoletes that carryset-1Bgene were associated with dehydration symptoms in children (P
<
0.001). The ShET-1B subunit is enterotoxin, and according to experimental models, it alters the transport of water and electrolytes into the small intestine [1, 9, 38, 39]. Our findings confirm ShET-1B subunit as a potentially aggravating factor for dehydration in shigellosis.


## 5. Conclusions

We conclude that this PCR typing was able to identify irrespectively virulence genes in wildShigella species, and our results showed vast genetic diversity ofShigella isolates. In addition, our study contributes to knowledge on particular symptoms of shigellosis associated with virulence genes, whose information about their roles are based on experimental models.



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*Source: 539697-2014-04-29.xml*