Flávia Corrêa BastosI,II; Edvaldo Carlos Brito LoureiroI
IDepartamento de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua, PA IIPrograma de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários, Universidade Federal do Pará, Belém, PA
ABSTRACT
INTRODUCTION: Shigella spp. are Gram-negative, nonsporulating, rod-shaped bacteria that belong to the family Enterobacteriaceae and are responsible for shigellosis or bacillary dysentery, an important cause of worldwide morbidity and mortality.
METHODS: We studied the antibiotic resistance profiles of 122 Shigella spp. strains (81 S. flexneri, 41 S. sonnei, 1 S. boydii) isolated from patients (female and male from 0 to 80 years of age) presenting diarrhea in different districts of the State of Pará, in the North of Brazil. The antibiotic resistance of the strains, isolated from human fecal samples, was determined by the diffusion disk method and by using the VITEK-2 system.
RESULTS: The highest resistance rate found was the resistance rate to tetracycline (93.8%), followed by the resistance rate to chloramphenicol (63.9%) and to trimethoprim/sulfamethoxazole (63.1%). Resistance to at least three drugs was more common among S. flexneri than S. sonnei (39.5% vs. 10%). Six (4.9%) strains were susceptible to all the antibiotics tested. All strains were susceptible to cefotaxime, ceftazidime, ciprofloxacin, nalidixic acid and nitrofurantoin.
CONCLUSIONS: High rates of multidrug resistance in Shigella spp. are a serious public health concern in Brazil. It is extremely important to continuously monitor the antimicrobial resistances of Shigella spp. for effective therapy and control measures against shigellosis.
Keywords: Shigella spp. State of Pará. Antimicrobial resistance. Diarrhea.
RESUMO
INTRODUÇÃO: Shigella spp. são bactérias Gram-negativas, não esporuladas, em forma de bastonete, pertencentes a família Enterobacteriaceae responsáveis pela shigelose ou disenteria bacilar, uma importante causa de mortalidade e morbidade mundial.
MÉTODOS: Foi estudado o perfil de resistência a antimicrobianos de 122 amostras de Shigella spp. (81 S. flexneri, 41 sonnei, 1 S. boydii) isoladas de pacientes (sexo feminino e masculino com faixa etária de 0 a 80 anos) com distúrbios gastrointestinais em diferentes municípios no Estado do Pará, Brasil. A resistência antimicrobiana das amostras isoladas de coprocultura, foi determinada pelo método de difusão em disco e pelo sistema Vitek II.
RESULTADOS: A maior resistência foi observada em relação à tetraciclina (93,8%), seguida de cloranfenicol (63,9%), e trimetoprimsulfametoxazol (63,1%). Multirresistência a pelo menos três antimicrobianos foi mais comum em S. flexneri comparada a S. sonnei (39,5% vs. 10%). Seis (4,9%) amostras foram sensíveis a todos antimicrobianos testados. Todas as amostras apresentaram sensibilidade a cefotaxima, ceftazidima, ciprofloxacina, ácido nalidixico e nitrofurantoína.
CONCLUSÕES: As altas taxas de multirresistência de Shigella spp. são um sério problema de saúde pública no Brasil. Sendo assim, torna-se extremamente importante um monitoramento contínuo da resistência antimicrobiana de Shigella spp. para uma terapia efetiva e medidas de controle contra shigelose.
Palavras-chaves: Shigella spp. Estado do Pará. Resistência antimicrobiana. Diarréia.
INTRODUCTION
Shigella spp. are Gram-negative, nonsporulating, rod-shaped bacteria that belong to the family Enterobacteriaceae. The bacteria are facultative intracellular pathogens that show a high specificity for primate hosts. Shigella spp., the human pathogens responsible for shigellosis, are highly infectious, even at low counts (102)1. Shigellosis is recognized by the World Health Organization as a major, global, public health concern2. It is responsible for morbidity and mortality in high risk populations, such as children under five years of age, senior citizens, toddlers in day-care centers, patients in custodial institutions, homosexual men, those affected by war and famine and patients with chronic disease (e.g., HIV), predominantly in developing countries3. Shigellosis is an acute intestinal infection the symptoms of which can range from mild, watery diarrhea to severe, inflammatory bacillary dysentery characterized by strong abdominal cramps, fever and stools containing blood and mucus4. The disease is usually self-limiting but may become life-threatening if patients are immunocompromised or adequate medical care is not available. A combination of oral rehydration and antibiotics leads to rapid resolution of infection. Unfortunately, Shigella spp. have become resistant to commonly used antibiotics, drastically reducing therapeutic possibilities, especially in children5-6 .
The genus Shigella includes four species: S. dysenteriae (serogroup A), S. flexneri (serogroup B), S. boydii (serogroup C), and S. sonnei (serogroup D). S. flexneri is the most common Shigella species in developing countries. In developed countries, S. sonnei is the most prevalent7. S. dysenteriae is implicated in epidemic disease outbreaks, the most severe form of dysenteryand the majority ofthefatal shigellosis cases8.
This study was conducted to identify the antimicrobial resistance profile of 122 strains of Shigella spp. (81 S. flexneri, 40 S. sonnei and 1 S. boydii) isolated during 1979-2009 from patients (male and female, from 0 to 80 years of age) who presented with diarrhea and were assisted at the Evandro Chagas Institute (IEC) in the State of Pará, Brazil.
METHODS
Bacterial strains
From January 1979 to July 2009, many samples of 122 Shigella strains (81 S. flexneri, 40 S. sonnei and 1 S. boydii) were isolated from the clinical specimens of different patients (male and female from 0 to 80 years of age) presenting with diarrhea and assisted at the Bacteriology and Mycology Department of the IEC. The patients included in the study were from nine different districts in the State of Pará: Abaetetuba, Ananideua, Belém, Benevides, Castanhal, Icoaraci, Marabá, Marituba and Moju. The isolates, which belong to the bacterial collection of the IEC, were stored at -70ºC in Luria Bertani (LB) broth with 15% glycerol and at environmental conditions.
Determination of antibiotic resistance
Ampicilin (4, 8, and 32µg/mL), amoxilin (4/2, 16/8, and 32/16µg/mL), ceftazidime (1, 2, 8, and 32µg/mL), ciprofloxacin (0, 5, 2, and 4µg/mL), nalidixic acid (8, 16, and 32µg/mL), nitrofurantoin (16, 32, and 64µg/mL), and trimethoptim/ sulfamethoxazole (0.5/9.5, 2/38, and 16/304µg/mL) were used to determine the resistance profiles of all Shigella strains studied using the VITEK-2 system (bioMérieux, Marcy I’Etoile, France). Thechloramphenicol (30µg), cefotaxime (30µg), gentamicin (10µg) and tetracycline (30µg) profiles of antibiotic resistance were determined by the diffusion disk method according to the Manual of the Clinical and Laboratory standards Institute (CLSI 2009)9. The standard reference strains of Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27,853 were used as controls throughout the study.
RESULTS
The resistances of the Shigella spp. strains to all the antimicrobials tested are shown in Table 1. A total of 115 (94.2%) were resistant strains. The highest resistance rate found was the resistance rate to tetracycline (93.4%), followed by the rate to chloramphenicol (63.9%) and to trimethoptim/sulfamethoxazole (63.1%). Sixty (49.2%) strains were multidrug-resistant (resistant to at least two antimicrobials).
A total of 7 (5.8%) strains were susceptible to all the antibiotics tested. All the strains were susceptible to cefotaxime, ceftazidime, ciprofloxacin, nalidixic acid and nitrofurantoin.
The antimicrobial resistance patterns of all the strains of Shigella spp. were compared to each other. Overall, 93.8% of the S. flexneri strains and 95% ofS. sonneistrains wereresistant to tetracycline. Seventy-five percent of the S. flexneri strains and 42.5% of the S. sonnei strains were resistant to chloramphenicol. Sixty-four percent of the S. flexneri strains and 62.5% of the S. sonnei strains were resistant to trimethoprim-sulfamethoxazole.
As shown in Table 2, only 39.5% of the S. flexneri strains were resistant to at least three of the antibiotics (AM, C, SXT) tested.
The most common antimicrobial resistance pattern observed was resistance to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole. Forty-five percent of the S. sonnei strains were resistant to only one antibiotic (trimethoprim-sulfamethozole). Resistance to at least three drugs (AM, C, SXT) was more common among S. flexneri than S. sonnei (39.5% vs. 10%).
DISCUSSION
It is well established that S. flexneri is the most commonly isolated species of the Shigella genus in developing countries, and its presence has been associated with inadequate sanitation; in contrast, S. sonnei predominates in developed countries but is predominantly involved in sporadic, common-source outbreaks. S. boydii was first detected in India, and up to now, it has rarely been found outside the Indian subcontinent10. The species of Shigella most frequently isolated in Brazil, mainly from children under 5 years of age, are S. flexneri and S. sonnei7. Research on the epidemiology and microbiological aspects of bacterial diarrhea in children from Salvador in the State of Bahia, Brazil, demonstrated that in the period from January of 2002 to December of 2003, 1,991 patients between 0 and 15 years of age had their stools cultured, of which 260 presented bacterial growth. Shigella spp. were the most common pathogens and were found in 141 (54.3%) cultures, of which 80.1% were identified as S. sonnei and 19.9% as S. flexneri11. The first study of gastroenteritis in the Amazonian area was accomplished in Santarém in the State of Pará, where 48% of the 320 patients with severe diarrhea were determined to have shigellosis12. In the period from 1979 to 1980, investigations were conducted at private clinics and in a public hospital in Belém, State of Pará, Brazil with the objective of establishing the role of enteropathogenic bacteria as the cause of acute diarrhea in children from 0 to 5 years of age. They found Shigella in 13% of the cases (S. flexneri [62%], S. sonnei [29%], S. boydii [6%] and S. dysenteriae [3%])13. In the present study, most of the strains identified were S. flexneri (66.4%) as well, followed by S. sonnei (32.8%) and S. boydii (0.8%).
The antibiotic resistance of Shigella spp. has been hindering the treatment of shigellosis, particularly in children5-6. A study conducted in the United States, involving 1,604 Shigella spp. strains isolated in the period from 1999 to 2002, revealed a high resistance (1,031/64%) to the antibiotics tested. The largest resistance (322/31%) was observed to the combination of ampicillin, estreptomicin and tetracycline, followed by ampicillin, estreptomicin and sulfametoxazol (149/14%) and ampicillin and estreptomicin (108/10%). Resistance to ampicillin in combination with cloranfenicol, estreptomicina, tetracycline and sulfametoxazole, was observed in 8% (85) of the strains5.
From June 2006 to February 2009, all patients with shigellosis reported in New York City were interviewed. Their Shigella isolates were tested for antimicrobial susceptibility to examine the level of resistance and to identify risk factors for resistance. Analysis was conducted on two groups distinguished by a large outbreak that was documented during the data collection period. Of the 477 non-outbreak patients, 333 (70%) reported taking an antibiotic for shigellosis. Thirty-six (11%) patients were treated with an antibiotic to which their Shigella infection was resistant. Among this group, high levels of antimicrobial resistance were detected to ampicillin (68%) and trimethoprim-sulfamethoxazole (66%)14.
Most of the S. flexneri strains isolated from cases of bacillary dysentery in Campinas, State of São Paulo, Brazil were resistant to more than one of the antibiotics tested, and 90% were resistant to at least three antibiotics. The antibiotics to which the bacteria were most commonly resistant were ampicillin (83.3%), chloramfenicol (70%), and tetracycline (80%). However, among the S. sonnei strains, resistance was observed to 2 (70%) or 3 (30%) antibiotics. Resistance to tetracycline was detected in 96.7% of the strains. Resistance to ampicillin (6.7%) was also detected among these strains7.
A study conducted on strains of different species of Shigella from cases of diarrhea in the State of Rondônia, Brazil in the period from March of 2000 to March of 2002 found high resistance to trimethoprim/sulfametoxazole and ampicillin. S. flexneri was the only species that presented multidrug resistance9.
Our study revealed that Shigella spp. isolated in Belém, State of Pará, Brazil were resistant mostly to tetracycline (93.4%), chloramfenicol (63.9%), trimethoprin/sulfametoxazole (63.1%) and ampicilin (43.4%). No resistance was found to cefotaxime, ceftazidima, ciprofloxacin or nalidixic acid.
Because of increasing antimicrobial resistance in Shigella spp., empirical treatment options are decreasing. Antimicrobial therapy for shigellosis requires knowledge of the antimicrobial resistance patterns of the Shigella spp. strains circulating locally. Physicians should be aware of the high multidrug resistance rates of Shigella spp., especially resistance to tetracycline and chloramfenicol. S. flexneri is still the most prevalent species in many Brazilian cities, and its higher antimicrobial resistance rate compared to that of S. sonnei is a cause for concern.
It is extremely important to continuously monitor the antimicrobial resistances of Shigella spp. for effective therapy and control measures against shigellosis.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
FINANCIAL SUPPORT
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Cnpq) and the Evandro Chagas Institute, Ananindeua/Pará/Brazil.
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Address to:
Dra. Flávia Corrêa Bastos
Deptº Bacteriologia e Micologia/IEC
Rodovia BR 316, Km 7 s/n
67030-000 Ananindeua, PA, Brasil
Phone: 55 91 3214-2122; Fax 55 91 3214-2128
e-mail: flavia_bastos@hotmail.com
Received in 26/01/2011
Accepted in 28/02/2011
