Antimicrobial Resistance of Shigella spp. isolated in the State of Pará , Brazil

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.

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 (10 2 ) 1 . Shigellosis is recognized by the World Health Organization as a major, global, public health concern 2 . 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 countries 3 . 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 mucus 4 . 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 children [5][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 prevalent 7 . S. dysenteriae is implicated in epidemic disease outbreaks, the most severe form of dysentery and the majority of the fatal shigellosis cases 8 .

Bastos FC and Loureiro ECB -
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% of S. sonnei strains were resistant 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. 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.

DISCUSSION
The most common antimicrobial resistance pattern observed was resistance to ampicillin, chloramphenicol and trimethoprimsulfamethoxazole. 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%).
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 subcontinent 10 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 children [5][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 strains 5 .
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 strains 7 .
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 resistance 9 .
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.