Epidemiological trends and patterns of antimicrobial resistance of Shigella spp. isolated from stool cultures in two different populations in Southern Israel

Southern Israel is inhabited by Bedouins, living in conditions similar to developing countries and Jews, living in conditions similar to developed countries. We determined the epidemiology of Shigella spp. in these populations. We retrospectively reviewed Shigella spp. stool isolations between 2005–2009. Overall, 3295 isolates were analyzed. S. sonnei was isolated in 2057/3295 (62.4%) and S. flexneri in 1058 (32.1%). S. sonnei was isolated in 1567/1707 (91.8%) from Jewish patients and S. flexneri in 931/1542 (60.4%) from Bedouin patients. Ampicillin resistance increased linearly from 217/373 (58.2%) in 2005 to 186/256 (72.7%) in 2009, (P < 0.001). Trimethoprim-sulfamethoxazole resistance decreased linearly from 328/373 (87.9%) in 2005 to 133/256 (51.9%) in 2009 (P < 0.001). Higher resistance of Shigella spp. to ampicilin and trimethoprim-sulfamethoxazole were found in Jewish patients: 1527/1706 (89.5%) versus 977/1542 (63.4%) (P < 0.0001), 1635/1706 (95.8%) versus 1026/1542 (66.5%) (P < 0.0001). The epidemiology of Shigella spp. infections can differ in populations residing in the same geographical area.     

Antimicrobial resistance of Shigella spp. from humans in Shanghai,China, 2004–2011

A retrospective study conducted on patients with diarrhea in Shanghai, China from 2004–2011, indicated that of 77,600 samples collected, 1,635 (2.1%) tested positive for Shigella. Species isolated included S. sonnei (1,066, 65.1%), S. flexneri (569, 34.7%), and S. boydii (3, 0.2%). Most of the Shigella isolates were found to be resistant to streptomycin (98.7%), trimethoprim (98.0%), ampicillin (92.1%), and nalidixic acid (91.7%). Additionally, many isolates were resistant to tetracycline (86.9%), trimethoprim + sulfamethoxazole (80.1%), sulfisoxazole (76.8%) and gentamicin (55.5%). Approximately 80% of the isolates were resistant to at least eight antimicrobial agents, 14% to at least ten antimicrobials tested and 10 isolates to fourteen antimicrobials, including sulfonamides, fluoroquinolones, tetracyclines, aminoglycosides and β-lactamases. Importantly, co-resistance to fluoroquinolones and the third- and fourth-generation cephalosporins was also identified. The high levels of resistance to antimicrobial agents commonly used in clinical medicine presents a great challenge to treating patients with shigellosis.     

Phylogenetic analysis and identification ofShigellaspp. by molecular probes

Oligonucleotide probes were used for identification ofShigellaand analysis of the relationship betweenShigellaspp. andEscherichia coli. Probe-based PCRs shown cross-reactions fromShigellatoE. coli. Probe-based 16S rRNA sequencing and phylogenetic analysis showed the four species ofShigella:Sh. dysenteriae,Sh. boydii,Sh. sonnei, andSh. flexneri, formed a cluster withE. coli. Shigella flexneriandSh. sonneiare even more similar toE. colithan to the other twoShigellaspecies. These results confirmed an earlier recommendation that the four species ofShigellaandE. colishould be classified as five sub-groups within a single species.     

Antimicrobial Resistance in Shigella flexneri and Shigella sonnei in Hong Kong, 1986 to 1995

Three hundred and thirty-three Shigella isolates obtained in 1986 to 1995 were tested for their susceptibilities to 19 antimicrobial agents. Nalidixic acid resistance had emerged in 59.6% of Shigella flexneri isolates during 1994 to 1995, with all tested resistant isolates having the mutation in gyrA encoding the Ser-83 alteration. Multiresistance (resistance to four or more agents) was more common in S. flexneri than in Shigella sonnei.     

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in João Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them.     

In Vitro Activity of Cinoxacin, Ampicillin, and Chloramphenicol Against Shigella and Nontyphoid Salmonella

The in vitro antibacterial activity of cinoxacin was compared with that of ampicillin and chloramphenicol against 26 strains of nontyphoid Salmonella and 44 strains of Shigella. Cinoxacin was found to have a lower minimal inhibitory concentration than ampicillin and chloramphenicol against all Salmonella and Shigella sonnei strains. Cinoxacin had minimal inhibitory concentrations similar to those of chloramphenicol but lower than those of ampicillin against Shigella flexneri and S. boydii strains.     

Antimicrobial Susceptibility and Mechanisms of Resistance in Shigella and Salmonella Isolates from Children under Five Years of Age with Diarrhea in Rural Mozambique

The antimicrobial susceptibility and mechanisms of resistance of 109 Shigella and 40 Salmonella isolates from children with diarrhea in southern Mozambique were assessed. The susceptibility to seven antimicrobial agents was tested by disk diffusion, and mechanisms of resistance were searched by PCR or colorimetric method. A high proportion of Shigella isolates were resistant to chloramphenicol (Chl) (52%), ampicillin (Amp) (56%), tetracycline (Tet) (66%), and trimethoprim-sulfamethoxazole (Sxt) (84%). Sixty-five percent of the isolates were multidrug resistant. Shigella flexneri isolates were more resistant than those of Shigella sonnei to Amp (66% versus 0.0%, P < 0.001) and Chl (61% versus 0.0%, P < 0.001), whereas S. sonnei isolates presented higher resistance to Tet than S. flexneri isolates (93% versus 64%, P = 0.02). Resistance among Salmonella isolates was as follows: Tet and Chl, 15% each; Sxt, 18%; and Amp, 25%. Only 3% of Salmonella isolates were resistant to nalidixic acid (Nal), and none to ciprofloxacin or ceftriaxone (Cro). Among Salmonella isolates, multiresistance was found in 23%. Among Shigella isolates, antibiotic resistance was related mainly to the presence of oxa-1-like β-lactamases for Amp, dfrA1 genes for Sxt, tetB genes for Tet, and Chl acetyltransferase (CAT) activity for Chl. Among Salmonella isolates, resistance was conferred by tem-like β-lactamases for Amp, floR genes and CAT activity for Chl, tetA genes for Tet, and dfrA1 genes for Sxt. Our data show that Shigella isolates are resistant mostly to the most available, inexpensive antibiotics by various molecular mechanisms but remain susceptible to ciprofloxacin, Cro, and Nal, which is the first line for empirical treatment of shigellosis in the country.Diarrhea disease is one of the main causes of childhood mortality worldwide. It is estimated that 17% of the 10.8 million deaths of children under 5 years of age worldwide is due to diarrhea, with developing countries being the most affected (6). Diarrhea can be caused by different agents, such as bacteria, parasites, and virus (1, 24, 35). The severity of the illness is mediated by different factors related to both the patient (nutritional status, presence of concomitant illness, and human immunodeficiency virus status) and the etiological agent (specific bacterial virulence and antimicrobial resistance). Salmonella spp. and Shigella spp. remain among the bacteria most frequently isolated from stool samples obtained from diarrhea patients, especially in rural areas from developing countries (24, 35). Salmonella spp. usually produce a self-limited illness, whereas infections due to Shigella are likely to be more severe.The management of acute diarrhea is based on replacement of fluids (8). However, antibiotics might be required for management of the most severe cases or cases involving malnourished children. With shigellosis, appropriate antimicrobial therapy can reduce the duration of fever and the period of shedding of the pathogens (26), which is relevant to transmission of the pathogen to susceptible contacts. At the Manhiça District Hospital (MDH), the combination of ampicillin (Amp) plus gentamicin used with children younger than 2 months of age and chloramphenicol (Chl) used alone with older children are the most available therapeutic options for treating bacterial infections (32), while nalidixic acid (Nal) is recommended for cases of dysentery.Until recently, Salmonella spp. were highly susceptible to the most commonly used antibiotics (34). However, in the last decade, the emergence of multidrug-resistant nontyphoidal Salmonella strains, including isolates resistant to quinolones, has been described worldwide (14, 33, 36). Also, the increase in the number of Shigella isolates resistant to most of the antibiotics available in countries where the choice of treatment is limited (14, 16, 21, 25, 36) represents an important health problem.The mechanisms of antimicrobial resistance are associated with intrinsic resistance, point mutations, and acquired or extrachromosomal resistance (31). A wide range of molecular mechanisms, such as the presence of β-lactamases, dihydrofolate reductase, Chl acetyltransferase (CAT) enzymes and many others (7, 10, 11, 22, 27), have been described. However, few studies have investigated molecular mechanisms of antimicrobial resistance among isolates from sub-Saharan Africa, due mainly to the limited number of laboratories and research facilities with an adequate infrastructure available on the continent (10, 21).Currently, data about antimicrobial resistance among diarrheagenic bacteria in Mozambique are scarce. This study describes the antimicrobial susceptibility and the molecular mechanisms of resistance in Salmonella and Shigella isolates from children with diarrhea in a rural hospital in Mozambique.     

A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive escherichia coli in human feces

A simple polymerase chain reaction (PCR) procedure using IS630-specific primers was developed as a general diagnostic probe to detect Shigella and enteroinvasive Escherichia coli (EIEC). However, IS630 and the other two previously reported molecular probes, ipaH and ial, cannot be used to differentiate among Shigella serotypes and EIEC strains that cause dysentery. The sensitivity of PCR protocol was determined to be 100–200 shigellae for each PCR reaction. An enrichment incubation would allow the detection of shigellae in stool samples with low bacterial concentration; i.e., <10⁴ CFU/gram. Serotype-specific primers derived from the rfc genes of different Shigella strains were used in PCR reactions to differentiate among Shigella serotypes in the laboratory, such as S. sonnei, S. flexneri, and S. dysenteriae 1. It was demonstrated further that the multiplex PCR system containing rfc-specific primers can efficiently identify the most prominent Shigella serotypes in raw stool samples of acute diarrheal patients.     

Immune responses to Shigella lipopolysaccharide and invasion plasmid antigens in Shigella infected Vietnamese patients

Immune responses were studied in 19 adult male Vietnamese patients with bacillary dysentery. Shigella flexneri was isolated in faeces from 17 patients (S. flexneri 2a (n = 11), S. flexneri 4a (n = 4), S. flexneri 3a (n = 1), and S. flexneri 3b (n = 1)) and S. dysenteriae 2 was isolated from 2 patients. Thirty healthy, adult males, without diarrhoea 3 months prior to the study, served as controls. The use of enzyme immune assay (EIA) showed that S. flexneri infected patients developed intestinal sIgA responses against S. flexneri Y lipopolysaccharide (LPS) and Shigella invasion plasmid antigens (Ipa), with peak values seen 2 weeks after the onset of diarrhoea. The sIgA titres were significantly higher (0.001 < P < 0.05 for anti-Shigella Ipa sIgA and 0.01 < P < 0.05 for anti-S. flexneri Y LPS sIgA) in the patients than in the healthy controls. Enzyme-linked immunospot (ELISPOT) analyses of peripheral blood mononuclear cells (PBMC) showed that S. flexneri infected patients developed significant antibody secreting cell (ASC) responses against S. flexneri Y LPS and Shigella Ipa (0.001 < P < 0.05), which peaked 1 week after the onset of diarrhoea. IgA-ASC predominated, followed by IgG-ASC, whereas IgM-ASC were least common. The number of Shigella Ipa-specific ASC was higher than the number of S. flexneri Y LPS-specific ASC. With use of EIA it was shown that S. flexneri infected patients had significantly (0.001 < P < 0.05) higher serum IgA and IgG titres than the healthy controls. All 17 patients had anti-S. flexneri Y LPS titres, and 11 patients had anti-Shigella Ipa titres exceeding the cut-off titres (mean ± 2S.D.) seen in the controls. The highest IgA titres were seen 1 week, and the highest IgG titres 2 weeks, after the onset of diarrhoea.     

High Prevalence of Antibiotic Resistance and Molecular Characterization of Integrons among Shigella Isolates in Eastern China

A total of 747 Shigella isolates were collected from hospitals in Jiangsu Province of China. Susceptibilities to antimicrobials and integrons were tested. A total of 78.3% of S. flexneri isolates and a total of 74.3% S. sonei isolates were resistant to at least three antibiotics. Of the Shigella isolates, 74.7% had integron I and 82.6% had integron II. The conjunction of the high prevalence of integrons in Shigella and high resistance to antimicrobials will lead to rapid dissemination of resistant genes in this region.     

Studies on experimental shigellosis. I. Shigella infections of normal mice

Experimental Shigella flexneri 3 infections produced in normal mice have been described. The passage of viable Shigella through the stomach of orally infected animals, and the persistence of the organisms in the intestine for periods of 5 to 7 days, with an increase in bacterial numbers, indicated that true infection was produced. Blood culture studies showed that a Shigella bacteriemia occurred after either oral or parenteral administration of Shigella to normal mice. Mice infected orally revealed mild, but consistent pathological changes, including mesenteric hemorrhage, liver and spleen hypertrophy, and occasional diarrhea.     

Shigella flexneri O-antigen-specific enzyme immunoassay: class-specific antibody titres against lipopolysaccharide antigens in healthy Vietnamese and Swedish populations

Sera collected from 397 healthy Vietnamese individuals, living in two districts in the surroundings of Hanoi, and 62 healthy Swedish individuals, living in the greater Stockholm area were analysed in enzyme-immunoassays (EIA) for their class-specific immunoglobulin titres against a panel of 10 O-antigen containing lipopolysaccharides (LPS) from Shigella flexneri, Shigella dysenteriae serotype I, Shigella sonnei and Salmonella serogroups A and B. The Vietnamese population was divided into age groups: 0·1–5 year, 1–2, 3–4, 5–9, 10–14 years, etc. Young Vietnamese children had low anti-S. flexneri LPS IgA, IgG and IgM titres which reached adult levels for IgA in the 10- to 14-year-old group, and for IgG and IgM in the 3- to-4-year-old group. The IgA, IgG and IgM titres were significantly higher (P < 0·001) in sera from Vietnamese individuals than in sera from Swedish individuals. The EIA titres against S dysenteriae serotype 1 and S. sonnei LPSs were higher in the Vietnamese than in Swedish population, but to a lesser extent than seen for S. flexneri. The titres against Salmonella serogroups A and B LPS antigens did not differ significantly in the study populations. These results suggest that the incidence of S. flexneri infections is high in Vietnam, followed in frequency by S. dysenteriae serotype I and S. sonnei infections. On the contrary the incidence of salmonellosis appears to be low.     

Serotypes,antibiotic resistance,and virulence genes of Salmonella in children with diarrhea

Background Salmonella is an important foodborne pathogen that causes acute diarrhea in humans worldwide. This study analyzed the relationships of serotypes and antibiotic resistance with virulence genes of Salmonella isolated from children with salmonellosis.MethodsSerological typing was performed using the slide‐agglutination method. The Kirby‐Bauer disk diffusion method was used to test antibiotic susceptibility. Twenty virulence genes were detected by PCR.Results Salmonella Typhimurium (21 isolates, 34.43%) and S Enteritidis (12 isolates, 19.67%) were the predominant species among the 61 isolates. Ampicillin resistance was most common (63.93%), and among the cephalosporins, resistance was most often found to cefotaxime, a third‐generation cephalosporin (19.67%). Among the 20 virulence genes, prgH, ssrB, and pagC were detected in all Salmonella isolates. In S Typhimurium, the detection rates of hilA, sipB, marT, mgtC, sopB, pagN, nlpI, bapA, oafA, and tolC were high. In S Enteritidis, the detection rates of icmF, spvB, spvR, and pefA were high. Nitrofurantoin resistance was negatively correlated with the virulence gene bapA (P = .005) and was positively correlated with icmF, spvB, spvR, and pefA (P = .012, .008, .002, and .005, respectively), The P values between all other virulence genes and antibiotic resistance were >.05.Conclusion Salmonella Typhimurium and S Enteritidis were the main serotypes in children with diarrhea in Hangzhou, China. Salmonella exhibited a high level of resistance to common antibiotics, and a high rate of bacteria carrying virulence genes was observed. However, no significant correlation was found between virulence genes and resistance to common antibiotics.     

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen.A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T_m = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C_q) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea.The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.     

Molecular Epidemiology of Escherichia coli Sequence Type 131 and Its H30 and H30-Rx Subclones among Extended-Spectrum-β-Lactamase-Positive and -Negative E. coli Clinical Isolates from the Chicago Region, 2007 to 2010

We assessed Escherichia coli ST131 and its H30 and H30-Rx subclones for virulence genes, antimicrobial resistance, and extended-spectrum beta-lactamase (ESBL) type. Although both subclones were associated with ESBL production, H30-Rx isolates had higher resistance scores and were associated specifically with CTX-M-15. Three virulence genes (iha, sat, and iutA) were more prevalent among H30 than non-H30 ST131 isolates. Thus, the H30 and H30-Rx subclones are more antimicrobial resistant and have virulence profiles that are distinct from those of non-H30 ST131 isolates.     

rho, a Small GTP-Binding Protein, Is Essential for Shigella Invasion of Epithelial Cells

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with α₅β₁ integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamster ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125^FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.     

Beta-Lactamase Activity in Shigella sonnei

The basis of ampicillin resistance in Shigella sonnei was analyzed for a number of isolates. Most isolates that were resistant to ampicillin and cephalothin failed to transfer resistance. Isolates resistant to cephalothin and ampicillin were inhibited by mecillinam, cefamandole, and cefoxitin. Six strains were selected for study. These differed on the basis of inhibition by 10 β-lactam antibiotics and on differences in β-lactamase activity. There was no correlation between the inhibitory concentration of antibiotic and the amount of antibiotic hydrolyzed. Use of sodium chloride and cloxacillin inhibition and acrylamide electrophoresis demonstrated that ampicillin resistance in Shigella is mediated by a number of different β-lactamases. Organisms can contain more than one type of β-lactamase.     

High frequency of strains multiply resistant to ampicillin, trimethoprim-sulfamethoxazole, streptomycin, chloramphenicol, and tetracycline isolated from patients with shigellosis in northeastern Brazil during the period 1988 to 1993.

The occurrence and antimicrobial resistance pattern of Shigella isolates obtained from persons in community and hospital-based studies of diarrhea and matched controls in northeastern Brazil were studied. The isolation rate of Shigella spp. from patients with diarrhea during 1988 to 1993 varied from 4.5% (26 of 575) for the urban community of Gonçalves Dias to 6.7% (12 of 179) and 5.9% (7 of 119) for Hospital Infantil and Hospital Universitário, respectively. Of the 55 Shigella isolates (45 from patients with diarrhea, 8 from controls, and 2 undetermined) 73% (40 of 55) were Shigella flexneri, 16% (9 of 55) were S. sonnei, 7% (4 of 55) were S. boydii, and 4% (2 of 55) were S. dysenteriae. Of 39 S. flexneri strains, over half were resistant to ampicillin, trimethoprim-sulfamethoxazole, or both. Over 64% were resistant to streptomycin, chloramphenicol, and tetracycline. Overall, 82% of all S. flexneri isolates were resistant to four or more antimicrobial agents tested. As elsewhere, in the northeast of Brazil, ampicillin and trimethoprim-sulfamethoxazole are no longer reliable for treatment of S. flexneri infection. Most Shigella strains were resistant to four or more antimicrobial agents. Nalidixic acid was still useful for treatment of infections due to S. flexneri.     

Hospital Pseudomonas aeruginosa: Surveillance of Resistance to Gentamicin and Transfer of Aminoglycoside R Factor

Most ampicillin-resistant Shigella are susceptible to cephalexin. Randomized treatment with cephalexin or ampicillin was given to 154 infants and children with acute diarrhea. Rectal swab cultures revealed Shigella in 42%, Salmonella in 6%, enteropathogenic Escherichia coli in 2%, and no pathogen in 50%. Cephalexin failed to eradicate Shigella after 5 days of treatment in 76% of patients as contrasted with 28% of ampicillin-treated patients with susceptible organisms. Shigella persisted in 78% of ampicillin-treated patients with resistant organisms. Diarrhea lasted more than 5 days in 43% of cephalexin-treated patients, in 56% of the ampicillin group with resistant organisms, but in only 9% of ampicillin-treated patients with susceptible organisms. The failure of cephalexin was due to the relatively high minimal inhibitory concentrations and minimal bacterial concentrations of 5 or 10 μg/ml and, although serum concentrations were twice the minimal bacterial concentration, they were not sufficient to demonstrate killing by the serum dilution method. In vitro susceptibility or resistance of Shigella to ampicillin correlated with clinical success or failure. Cephalexin is not a suitable drug for treatment of shigellosis in patients with ampicillin-resistant organisms.