Clinical Isolates Of Shigella Research Articles

Molecular characterization of multidrug-resistant Shigella spp. of food origin

Shigella spp. are the causative agents of food-borne shigellosis, an acute enteric infection. The emergence of multidrug-resistant clinical isolates of Shigella presents an increasing challenge for clinicians in the treatment of shigellosis. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin, however, to date, no such characterization has been reported for Shigella spp. of food origin. In this study, we characterized the genetic basis of multidrug resistance in Shigella spp. isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Twenty-four out of 27 Shigella isolates (88.9%) showed multidrug resistance phenotypes to at least three classes of antimicrobials. The multidrug-resistant Shigella spp. were as follows: Shigella flexneri (66.7%), Shigella sonnei (18.5%), and Shigella dysenteriae (3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes. Our results indicated that 11.1% and 74.1% of isolates were positive for class 1 and class 2 integrons, respectively. Beta-lactamase-encoding genes were identified in 77.8% of isolates, and plasmid-mediated quinolone resistance genes were identified in 44.4% of isolates. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. To the best of our knowledge, this is the first report of the molecular characterization of antibiotic resistance in Shigella spp. isolated from food.

Plasmid-Mediated Sulfamethoxazole Resistance Encoded by the sul2 Gene in the Multidrug-Resistant Shigella flexneri 2a Isolated from Patients with Acute Diarrhea in Dhaka, Bangladesh

In this study, mechanisms of plasmid-mediated sulfamethoxazole resistances in the clinical strains of multi-drug resistant (MDR) Shigella flexneri 2a were elucidated for the first time in Bangladesh. From 2006 to 2011, a total of 200 S. flexneri 2a strains were randomly selected from the stock of the Enteric and Food Microbiology Laboratory of icddr,b. Antimicrobial susceptibility of the strains showed 73%, 98%, 93%, 58%, 98%, 64% and 4% resistance to trimethoprim-sulfamethoxazole, nalidixic acid, ampicillin, erythromycin, tetracycline, ciprofloxacin and ceftriaxone respectively. Plasmid profiling revealed heterogeneous patterns and interestingly, all the trimethoprim-sulfamethoxazole resistant (SXTR) strains yielded a distinct 4.3 MDa plasmid compared to that of the trimethoprim-sulfamethoxazole susceptible (SXTS) strains. Curing of this 4.3 MDa plasmid resulted in the susceptibility to sulfamethoxazole alone suggesting the involvement of this plasmid in the resistance of sulfamethoxazole. Moreover, PCR analysis showed the presence of sul2 gene in SXTR strains which is absent in SXTS strains as well as in the 4.3 MDa plasmid-cured derivatives, confirming the involvement of sul2 in the resistance of sulfamethoxazole. Furthermore, pulsed-field gel electrophoresis (PFGE) analysis revealed that both the SXTR and SXTS strains were clonal. This study will significantly contributes to the knowledge on acquired drug resistance of the mostly prevalent S. flexneri 2a and further warrants continuous monitoring of the prevalence and correlation of this resistance determinants amongst the clinical isolates of Shigella and other enteric pathogens around the world to provide effective clinical management of the disease.

Molecular characterization of extended spectrum β-lactamase (ESBL) producing Shigella species isolated from patients with bacillary dysentery in Iraq

Background: Extended-spectrum β-lactamases (ESBLs), including cefotaximases (CTX-M), mediate resistance to extended spectrum cephalosporins and significantly compromise the treatment tools for Shigellosis.
 Objective: To determine the ESBLs production by Shigella spp. and its role in the resistance to third generation cephalosporins and to determine the occurrence of plasmid- borne blaCTX genes in ESBLS Shigella isolates by Multiplex PCR.
 Methods: Susceptibility of 59 clinical Shigella isolates was tested by disk diffusion method to six antimicrobial agents. Presence of ESBLs was established by the combination disk method. Minimum inhibitory concentration of β-lactams was determined by agar dilution method for resistant isolates, and then ESBL producers were subjected to plasmid extraction and PCR experiments.
 Results: Overall isolates had high rate of resistance to Ampicillin (84.7%), Tetracycline (84.7%), Co-trimoxazole(72.9%) and Cefotaxime(69.5%). Moderate to Ceftriaxone (52.5%) and low rate of resistance to Ceftazidime(30.5%). The MIC value of resistant isolates of S. sonnei and S. flexneri for CTX and CRO ranged from (64-512 μg/ml) for both, and (32-256 μg/ml) for CAZ, whereas S. dysenteriae exhibit only high level resistance MIC (256 μg/ml) for CTX, low level resistance (64 μg/ml) for CRO and complete susceptibility to CAZ. 31 of 41 (75.6 %) of Shigella spp. were β-lactamase producers, of these (82.6%) were S. flexneri, (68.8%) S. sonnei, and one S. dysenteriae isolate. The results of PCR amplification of bla CTX gene groups showed that blaCTX-M genes were identified in (74.19%) ESBLs isolates distributed as 14(45.16%) S.flexneri isolates, 9(29.03%) S. sonnei, while no gene was detected in ESBLs S. dysenteriae isolate. The prevalence of bla CTX-M I, bla CTX-M II (TOHO 1) and blaCTX-M III, were ( 54.8%, 16.1% and 3.2% ) respectively. Non of the isolates carried more than one type.
 Conclusion: The occurrence of plasmid- borne blaCTX genes in ESBLS Shigella isolates are increasing in Iraq with co-resistance to some other classes of antibiotics

Detection of plasmid-mediated quinolone resistance determinants and the emergence of multidrug resistance in clinical isolates of Shigella in SiXian area, China

A total of 123 Shigella isolates were collected from SiXian area in Anhui, China. Screening was carried out by polymerase chain reaction (PCR) amplification of plasmid-mediated quinolone resistance (PMQR) determinants. Different β-blactamases genes, plasmid-borne blaAmpC, 16S rRNA methylase genes, integrons, and mutations in quinolone resistance-determining regions were analysed by PCR for the PMQR-positive isolates.

Antibacterial Activities of Polyethylene Glycol, Tween 80 and Sodium Dodecyl Sulphate Coated Silver Nanoparticles in Normal and Multi-Drug Resistant Bacteria

Antibacterial activity of silver nanoparticles coated with different functionalizing agents i.e., polyethylene glycol, tween 80 and sodium dodecyl sulphate were evaluated on both normal and multi-drug resistant strains of bacteria. Under the same reaction conditions, these functionalizing agents were added separately to coat silver nanoparticles. Among these, polyethylene glycol coated nanoparticles were most effective in killing all the bacterial strains which includes Escherichia coli DH5a, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and multi-drug resistant clinical isolates of Shigella spp. (flexneri, boydii, sohnea) and Vibrio cholerae. The minimum inhibitory concentration of polyethylene glycol coated silver nanoparticles was also less compared to the other two sets of nanoparticles. Consistence with that polyethylene glycol coated nanoparticles produced more intracellular reactive oxygen species in bacteria. Moreover, when human cell lines MCF7 and Chang Liver were incubated in presence of these nanoparticles for 18 h with same concentrations as used for bacteria, no toxicity was observed. But significant increase in cell killing was observed with longer incubation time. Thus our present investigation implicates the potential therapeutic use of silver nanoparticles as antibacterial agent particularly the polyethylene glycol coated one.

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins.

BackgroundThe entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.MethodsThe mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.ResultsThe clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucin<guinea pig colonic mucin< Human colonic mucin. Invasion of Shigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.ConclusionsOur study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.

Prevalence of the plasmid-mediated quinolone resistance determinants among clinical isolates of Shigella sp. in Andaman &amp; Nicobar Islands, India

This study was carried out to find the prevalence of various plasmid-mediated quinolone-resistant (PMQR) determinants among the quinolone-resistant clinical isolates of Shigella sp. from paediatric patients in Andaman & Nicobar Islands. A total of 106 quinolone-resistant Shigella isolates obtained from paediatric patients during hospital-based surveillance from January 2003 to June 2010 were screened for the presence of various PMQR determinants. Of 106 isolates, 8 (7.5%) showed the presence of aac (6')-Ib-cr and 3 (2.8%) harboured the qnrB genes with 2 (1.9%) of these isolates showing the presence of both. All the 9 isolates had uniform mutations in gyrA (S83L) and in parC (S80I). The prevalence of fluoroquinolone-acetylating aminoglycoside acetyltransferase {aac (6')-Ib-cr} gene is higher than qnrB gene among the clinical Shigella isolates. These PMQR determinants were detected in the Shigella isolates obtained from 2008-2010, indicating that it happens in a stepwise manner following the multiple mutations in quinolone resistance-determining regions increase or extend resistance to quinolones or fluoroquinolones. The prevalence of these genes are of grave concern as it may be horizontally transferred to other human pathogenic bacteria and can lead to therapeutic failure as a consequence of antimicrobial resistance, not only for the islands but also for the entire south-east region. The results obtained should encourage further studies on the implications of the presence, distribution, association and variation of these determinants in our quest for understanding PMQR.

Serotypes and extended-spectrum β-lactamase types of clinical isolates of Shigella spp. from the Zhejiang province of China

To understand the distribution of the serotypes of Shigella spp. and to investigate the drug-resistant genes of extended-spectrum β-lactamases (ESBLs) in Shigella in Zhejiang province, we have collected clinically isolated Shigella isolates from 2 hospitals in Zhejiang province during August to December of 2006. There are 11 Shigella flexneri and 13 Shigella sonnei isolates, respectively. Among the 11 S. flexneri, 9 are serotype F4 and the remaining 2 are F1a and F2a. Antimicrobial susceptibility tests revealed that 20.8% of the isolates were resistant to cefotaxime and 20.8% of the isolates were intermediate to cefotaxime. Isoelectric focusing demonstrated that the ESBL-positive isolates and their transconjugants produce a single β-lactamase with a p I of 7.9 or 9.0. DNA sequence analysis demonstrated that they harbor either CTX-M-14 or CTX-M-15 gene. Pulsed-field gel electrophoresis analysis showed that 9 F4 S. flexneri isolates belong to the same clone and 13 S. sonnei isolates from different regions in Zhejiang province belong to different subclones of the same clone. In summary, resistance and reduced susceptibility to β-lactam antibiotics were mainly caused by the production of CTX-M-type ESBLs. This is the first report of CTX-M-15–type ESBLs in S. sonnei in China.

Antimicrobial resistence of &lt;i&gt;Shigella&lt;/i&gt; species isolated during 2004 and 2005 from selected sites in Zimbabwe.

To determine the predominant serotype and antibiotic sensitivity pattern of Shigella isolates during 2004 and 2005 in Zimbabwe. Cross sectional study. National Microbiology Reference Laboratory (NMRL), Harare, Zimbabwe. 259 clinical isolates of Shigella species isolated during 2004 and 2005 in Zimbabwe were studied. These samples had been referred to the NMRL for further testing. Serotype and antibiotic sensitivity pattern of Shigella species. Of the 259 clinical isolates of Shigella tested the following species were serotyped; 141 (54.4%) were S. flexneri; 70 (27%) S. sonnei; 38 (14.7%) S. dysenteriae and 10 (3.9%) S. boydii. About 4% of all Shigella isolates tested showed full sensitivity to commonly used antibiotics, 20.8% were resistant to one antibiotic only while 75.3% were resistant to at least two antibiotics. The most common resistance among Shigella species was to cotrimoxazole (89%), tetracycline (73%), ampicillin (49%) and chloramphenicol (41%). High susceptibility among Shigella species was observed to nalidixic acid (86%), ciprofloxacin (99%) and ceftazidine (99%). There was a low drug resistance of Shigella species to nalidixic acid, a drug of choice in Zimbabwe, except among Shigella dysenteriae type 1 strains. Continuous monitoring of the susceptibility patterns of Shigella species is important in order to detect the emergence of drug resistance and to update guidelines for antibiotic treatment in shigellosis.

CTX-M-3 Type Beta-Lactamase Producing &lt;i&gt;Shigella sonnei&lt;/i&gt; Isolates from Pediatric Bacillary Dysentery Cases

In this study, 153 clinical isolates of Shigella were screened for extended-spectrum beta-lactamase (ESBL) production by double-disk synergy test. After being confirmed by the combined disk-test and inhibitor-combined E-test strips, all positive isolates were tested for the bla gene by PCR and the enzymes by isoelectric focusing. DNA sequencing of PCR products revealed that five Shigella sonnei isolates had CTX-M-3 type ESBL enzymes. All of them were resistant to ampicillin, sulfamethoxazole and cefotaxime but susceptible to ofloxacin and ceftazidime. All isolates displayed identical patterns in pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. This study reports multidrug-resistant (MDR) S. sonnei isolates producing CTX-M-3 type ESBL from successive pediatric bacillary dysentery patients, indicating widespread and rapid spread of CTX-M type ESBL in Shigella spp. To counter this emerging threat to public health, the surveillance of CTX-M type beta-lactamases should be considered, together with measures designed to prevent outbreaks of MDR Shigella in the community.

Identification of an atypical form of 30 kDa SOD—a possible virulence factor in clinical isolates of Shigella spp.

Reviews on the pathogenic mechanisms of Shigella species show a lacunae in the understanding of the bacterial antioxidant defense system and its regulations. This study was done to investigate the regulation of expression of antioxidant enzymes in clinical isolates of Shigella species, under various growth conditions. The in vitro expression of superoxide dismutase in the clinical isolates of Shigella spp., is modulated by both endogenous and exogenous factors. During aerobic and iron repleted growth conditions, the expression of the MnSOD and FeSOD enzymes were higher, and an atypical SOD was also expressed. However, under anaerobic growth conditions and in plasmid-cured strains, the antioxidant enzyme activities were decreased and the atypical SOD was not expressed. Absence of the atypical form of SOD may be due to the low oxygen environment. Plasmid-encoded factors may also play a role in the expression of this SOD, which had a molecular weight of approximately 30 kDa. In the rat ileal loop ligation assay, mild lesions were observed only in the intestinal microvilli of rats injected with plasmid-cured strains of Shigella spp., suggesting that plasmid-encoded factors, including those that regulate the expression of the atypical SOD, are essential for the virulence of Shigella spp.

Genetic analysis of antibiotic-resistance determinants in multidrug-resistant Shigella strains isolated from Chilean children

A total of 162 clinical isolates of Shigella collected from children in a semi-rural community of Chile were examined for the presence of genetic determinants of resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim. Ampicillin resistance was most frequently associated with the presence of bla(OXA) in S. flexneri and with bla(TEM) in S. sonnei. The bla(OXA) gene but not bla(TEM) was located in class 1 integrons. The dhfrIa gene encoding for resistance to trimethoprim was associated to class 2 integrons and detected exclusively in S. flexneri, whereas dhfrIIIc was found in all S. sonnei strains and in 10% of the S. flexneri isolates. Cat, coding for choramphenicol resistance, and bla(OXA) genes were located in the chromosome in all cases, whereas tetA gene, coding for tetracycline resistance, and bla(TEM), dhfrIa and dhfrIIIc genes were found either in the chromosome or in conjugative plasmids. Our results show a heterogenous distribution of antibiotic-resistance determinants between S. flexneri and S. sonnei.

Prevalence of the genes for shigella enterotoxins 1 and 2 among clinical isolates of shigella in Israel.

Two enterotoxins, shigella enterotoxin 1 (SHET1) and shigella enterotoxin 2 (SHET2) have been recently characterized and are believed to play a role in the clinical manifestation of shigellosis. One hundred and twenty-one isolates of Shigella spp. of 13 different serotypes and variants and 10 isolates of enteroinvasive Escherichia coli (EIEC) isolated in Israel, were examined by polymerase chain reaction for the presence of SHET1 and SHET2 genes. SHET1 was only prevalent among isolates of S. flexneri 2a while SHET2 was found in all the serotypes that were tested except for several isolates of S. flexneri 1b that lost their virulence plasmid during storage. In addition, we found that the S. flexneri 2a vaccine strain T-32 Istrati contains the gene encoding for SHET1 but not that encoding for SHET2, suggesting that the latter is located within a large deletion occurring in the 140 Mda plasmid of this S. flexneri 2a non-invasive vaccine strain.

Identification of Shigella serotypes by restriction of amplified O-antigen gene cluster

Due to the scarcity of distinctive biochemical reactions for differentiation of Shigella-Escherichia coli, antigenic analysis has long been used for identification and typing of Shigella isolates. Nevertheless, several intra- and interspecific cross-reactions have been reported to disturb serotyping assays. Shigella serotyping is also occasionally affected by the transition from the smooth (S) form to the rough (R) form. Thus, there is a need for the development of novel robust and discriminating methods for Shigella identification and typing. Characteristically, all genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella, and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and gene gnd, the conserved flanking sequences upstream and downstream of O-antigen gene clusters, were used to amplify the O-antigen gene cluster of representative strains of each Shigella serotype. A unique, amplified fragment was generally observed for each serotype (size ranging from 6 kbp to 17 kbp). Clearly identifiable and reproducible patterns were obtained for each serotype after MboII digestion of the products, except for S. boydii 12 which showed two distinct patterns, and S. flexneri serotypes 1 to 5 and X and Y which showed a single pattern. A database was built with the Taxotron package allowing automated identification of clinical Shigella isolates to all known serotypes.

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria.

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli. We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA. These shuA-positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA, so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae. The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA. The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA-positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae, indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae, and it is located in the same relative position on the chromosome.

Characterization of the Stable Maintenance of theShigella flexneriPlasmid pHS-2

pHS-2 is a 3-kb plasmid originally isolated fromShigella flexneriinfections associated with reactive arthritis in humans. This plasmid is stably maintained in many clinical isolates ofShigella flexneri.The nucleotide sequence of this plasmid displays two closely linked regions that may play a role in the maintenance of this plasmid. One region consists of a 250-bp locus showing a significant homology to the ColE1cersite. The results indicate that thecer-like site of pHS-2, like the ColE1cersite, acts as arecA-independent, site-specific recombination site involved in the resolution of multimers, requiring the presence of the host-encoded factors ArgR, PepA, XerC, and XerD. The second region consists of a 36-kDa open reading frame involved in generating resistance to the bactericidal effect of complement, which confers a selective advantage to cells containing this sequence. The results also indicate that pHS-2 can replicate in another species of Enterobacteriaceae (Escherichia coli) and is mobilized by the F plasmid.

Differences among Shigella spp. in susceptibility to the bactericidal activity of human serum.

Clinical isolates of Shigella spp. were examined for their susceptibility to human serum. The susceptibility of the strains to immune and nonimmune human serum was dependent upon the size of the bacterial inoculum and the concentration of serum. There were differences among Shigella spp. in susceptibility to human serum: S. sonnei strains were the least susceptible, strains of S. boydii and S. flexneri serotype 6 were intermediate, and those of S. flexneri other than serotype 6 and S. dysenteriae were the most susceptible. Experiments in which heat-treated (56 degrees C for 30 min, or 50 degrees C for 20 min) serum was used, and analysis of activation of complement by lipopolysaccharides (LPS) from each Shigella sp., suggested that LPS composition, especially the O antigen polysaccharide chains, contributes to the differences among Shigella spp. in susceptibility to human serum.

Ampicillin Resistance of Shigella sonnei

The ampicillin resistance of clinical isolates of Shigella sonnei is due to beta-lactamase production. Two kinds of resistance are found: low level, nontransmissible; and high level, tranmissible. The nontransmissible type of resistance results from a chromosomal mutation which increases the production of a beta-lactamase that hydrolyses cephalosporins relatively rapidly and gives cephalothin resistance. The transmissible type of resistance is due to an R factor mediating the synthesis of a different beta-lactamase that does not significantly hydrolyze cephalosporins or confer cephalothin resistance. One clinical isolate is shown to possess simultaneously both these mechanisms of ampicillin resistance.