Occurrence of CRISPR-Cas genes and lack of association with antibiotic resistance in Shigella isolates collected from patients with diarrhea in Ahvaz, southwest Iran

Objective: To investigate the antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran. Methods: In this study, 1 530 stool samples were collected from children under 15 years with diarrhea referred to teaching hospitals in Ahvaz and Abadan, southwest Iran. Shigella spp. were identified by standard biochemical tests and PCR. The antibiotic resistance pattern of all Shigella isolates was determined by the disk diffusion method and minimum inhibitory concentration (MIC) by E-test. Results: Of 1 530 stool samples, 91 (5.9%, 91/1 530) were positive for Shigella spp. the most common Shigella isolates were Shigella flexneri 47 (51.6%, 47/1 530). Antibiotic susceptibility tests showed that the highest antibiotic resistance was related to trimethoprim-sulfamethoxazole (87.9%, 80/91) and ampicillin (86.8%, 79/91). Multiplex PCR results revealed that 56% and 86.9% of Shigella isolates carried integron class I and integron class II genes, respectively. None of the isolates included the integron class III gene. Conclusions: The high prevalence of multi-drug resistance in Shigella isolates in our area increases the concerns about dissemination of the antibiotic-resistant isolates in this bacterium.

Shigellosis is a major public health problem, and increasing antimicrobial resistance has complicated its treatment. We report isolation frequency, plasmid profiles and antimicrobial resistance of Shigella subtypes in Kolkata, India, from a prospective hospital-based study. Fresh stool or rectal swabs were collected from children (<5 years) attending the Diarrhea Treatment Unit of a governmental paediatric referral hospital in Kolkata. Samples were processed following standard methods over a 7-year period from January 2001 to December 2007. Of 4478 samples collected, 516 (11.5%) were positive for Shigella spp. S. flexneri (312; 6.9%) was the most frequently isolated serogroup, ranking before S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Although 10 subtypes of S. flexneri were identified, the common ones circulating locally were S. flexneri 2a (179), S. flexneri 6 (38) and S. flexneri 3a (36). Knowledge of Shigella subtypes is important for vaccine development. The majority of Shigella isolates (81.0%) were multidrug (two or more antimicrobial classes) resistant and showed high minimum inhibitory concentration (MIC) with commonly used drugs like ampicillin, tetracycline, co-trimoxazole and nalidixic acid. Emergence of fluoroquinolone (FQ)-resistant S. dysenteriae type 1 (100.0%) in 2002-2003 was followed by frequent isolation (>25.0%) of FQ-resistant S. flexneri 2a, and S. flexneri 3a in 2004, which restricted use of fluoroquinolones for treatment. A number of smaller plasmids (<20 kb) with distinct patterns have been observed for several years in predominant subtypes. Long-term surveillance of Shigellae and their antimicrobial resistance are mandatory in endemic areas to formulate treatment policy until any suitable candidate vaccine is available to control the disease.

Introduction: This study was conducted to determine the frequenc y and pattern of antimicrobial susceptibility of Shigella spp. isola ted from pediatric hospital in two different time periods between March 1995 to March 1999 and March 2009 to March 2013 in North-West of Iran. Methods: The stool specimens were collected and examined fo r shigellosis by biochemical tests, and antibiogram was conducted according to C linical and Laboratory Standards Institute protocol. One hundred and thirty-nine Shigella spp. isolated from year 1995 to 1999 and 38 Shigella spp. isolates collected from year 2009 to 2013 and examined for serotyping and antibiotic resistance pattern. Results: According to serotyping results Shigella flexneri isolated in 98.6% of isolates in the first time period, followed by Shigella boydii and Shigella sonnei (0.7%) but in the second time period just 47.3% were S. flexneri and 39.5% were S. sonnei, 7.9% were S. boydii and 5.3% of isolates were Shigella dysenteriae. Results indicated significantly increase in resistance to ceftizoxime, chloramphenicol, and ami kacin (P = 0.004, 0.010, and 0.004 respectively), also, in Shigella isolates isolated in the second time period showed an increase in multidrug resistant (MDR) isolate and frequency of MDR isolates increased to 95.0% in the second time period. Conclusion: We are facing with the increase in resistance to a ntibiotics in Shigella spp. especially MDR isolates. These results showed changing pattern of resistance in Shigella isolates and needs for planning and design antibiot ics stewardships for controlling Shigellosis, especially in pediatric hospitals. Article info

Owing to the scarce evidence about the multidrug-resistant (MDR) beta-lactamase-producing Shigella isolates in Iran, this study aimed to evaluate the occurrence of extended-spectrum beta-lactamases (ESBL) and AmpC β-lactamases in Shigella species collected in the southwest of Iran. This study was conducted on Shigella species isolated from stool samples of pediatric patients aged less than 15years suffering from diarrhea. These isolates were identified by bacteriology tests, serotyping, and polymerase chain reaction (PCR). The antibiotic resistance was determined by disc diffusion. The production of ESBLs and AmpC was investigated by phenotypic confirmatory tests and PCR. In total, 79 Shigella isolates, including 46.8% (n = 37) of S. flexneri and 53.2% (n = 42) of S. sonnei, were isolated, respectively. The most effective antibiotic was imipenem with 93.7% of susceptibility followed by ampicillin (29.1%), and cotrimoxazole (30.4%).The resistance rates of ceftriaxone, ceftazidime, and cefotaxime were 41.8%, 34.2%, and 41.8%, respectively. Also, a total of 57 (72.2%) isolates showed MDR profiles. The phenotypic tests showed that 43.0% (34/79) of isolates can produce ESBLs, and no one was positive for ApmC. The frequency of blaTEM and blaCTX-M were 30.4% and 32.9%, respectively, while the blaPER, blaSHV, and AmpC genes were not detected. The ESBL-producing isolates had a significant (p-value˂0.05) resistance rate against ceftriaxone, ceftazidime, cefotaxime, cefepime, erythromycin, and amikacin. The significant prevalence of MDR Shigella isolates harboring ESBL genes highlights the need for effective surveillance measures to prevent the more spread of drug resistance among species.

Background: Shigella is one of the diarrhoea causing organisms found in HIV positive patients. But so far, the pattern of diarrhoeal agents caused by Shigella in AIDS patients has not been determined. Objective: This study is thus aimed at determining the prevalence, antimicrobial susceptibility and resistance of Shigella isolates in HIV positive subjects. Methods: All stool samples taken from the subjects of this study were plated on the MacConkey agar and incubated at 35-37oC for 24 or 48 hrs. Biochemical and antimicrobial sensitivity testing were carried out by using the standard methods. Results: Out of the 391 subjects included in the study, 199(63.8%) HIV seropositive and 113 seronegative patients had acute and chronic diarrhoea while 79 were HIV seropositive without diarrhoea. Of the 27 (8.7) Shigella isolates taken from the diarrhea patients, 11 (3.5%) were from HIV positive subjects. All Shigella isolates were found to be sensitive against norfloxacin (100%), gentamicin (97%), polymyxin B (97%) and kanamycin (93%). The most frequent resistance observed was to chloramphenicol (62%), tetracycline (86%) and ampicillin (100%). The frequency of resistance of Amp, Sex, Ch, TTc was found to be very high when compared with other patterns of resistance. Conclusion: The high proportion of HIV seropositive patients who had diarrhea in the absence of identified Shigella strains strongly indicates the existence of other diarrhoeagenic agents or mechanisms. Detailed investigation is important to get comprehensive information for better treatment of diarrhoea in HIV /AIDS patients. According to this finding, norfloxacin, gentamicin, polymyxin B, kanamycin and nalidixic acid might be used as drugs of choice for empirical treatment. On the other hand, amplicilin, tetracycline and chloramphenicol may not be used as the drugs of choice for the treatment of Shigella infection unless culture and sensitivity tests are done prior to treatment. The Ethiopian Journal of Health Development Vol. 20 (2) 2006: 99-105

Diarrhea is one of the important public health problems in developing countries. Salmonella and Shigella species are the major bacterial causal agents of diarrhea. The increasing burden of antimicrobial resistance is posing difficulty in the treatment of these pathogens. This study aimed to assess the occurrence of Salmonella and Shigella in the feces of diarrheic patients receiving health services in Addis Ababa, Ethiopia, and to determine their antimicrobial susceptibility profile. A cross-sectional study involving 13 health centers was conducted where 428 diarrheic patients were recruited. Standard microbiology techniques were used to isolate Salmonella and Shigella from stool samples. In addition, Salmonella isolates were confirmed by polymerase chain reaction (PCR). The Kirby-Bauer disc diffusion method was employed to assess susceptibility to 11 antimicrobials for each of the Salmonella and Shigella isolates. The prevalence of Salmonella and Shigella spp. among diarrheic patients was 8.4%; n = 36 and 5.6%; n = 24, respectively. Thirty (83.3%) of Salmonella isolates were susceptible to all antimicrobials tested, whereas 4 (10.8%) of isolates were resistant to 2 or more antimicrobials and 2 (5.6%) were multidrug resistant. Resistance to ampicillin was recorded in only one (2.7%) of Salmonella isolates; however, resistance to ampicillin was recorded in 12 (50%) of the Shigella isolates. Half of the Shigella isolates (n = 12) were resistant to 2 or more antimicrobials while 5 (20.8%) of them were resistant to 3 or more antimicrobials. The overall rate of resistance to antimicrobials was more common in Shigella compared to Salmonella isolates. In conclusion, Salmonella and Shigella were isolated from the feces of diarrheic patients, with a higher rate of antimicrobial resistance in Shigella isolates, which could make the treatment of shigellosis challenging. Therefore, increasing hygienic practices during food preparation to reduce the burden of Salmonella and Shigella infection and prudent use of antimicrobials are recommended to limit the spread of antimicrobial resistant strains.

This study was set up to assess the patterns of antimicrobial susceptibility and mutations in acrA-tolC genes of Shigella isolates and its association. One hundred and three isolates of Shigella spp. were tested to evaluate the antimicrobial susceptibility and mutation on acrA and tolC genes. The antimicrobial resistance profiles were: tetracycline (99%), ampicillin (85.4%), chloramphenicol (83.5%), trimethoprim (85.4%), ciprofloxacin (25.2%), cefazolin (3.9%), cefotaxime (3.9%), and gentamicin (2.9%). The rate of mutation in tolC was 90% (S. flexneri) and 30.8% (S. sonnei). Shigella flexneri isolates were more resistant than those of Shigella sonnei to chloramphenicol (97.8 versus 0.0%, p < 0.001) and ciprofloxacin (27.8 versus 7.7%, p = 0.106). High frequency of mutation was found in gene tolC (82.5%), but relatively less in acrA (22.3%). Shigella flexneri isolates were more mutated in tolC gene than S. sonnei (90 versus 30.8%, p < 0.001). Our study suggested that mutation of acrA and tolC may play major role in multiple antimicrobial resistance in Shigella spp. Shigella isolates are emerging which are resistant to first and third generation cephalosporin like cefazolin and cefotaxime, which is a matter of concern in terms of shigellosis treatment.

INTRODUCTION: Shigellosis still remains a public health problem in developing countries because of poverty, poor sanitation, personal hygiene and poor water supply. Antimicrobial therapy for shigellosis reduces the duration and severity of the disease and can also prevent potentially lethal complications. However, over the past few decades Shigella spp. has become resistant to most of the widely used antimicrobials. This study assessed the patterns of antimicrobial susceptibility and mutations in marA and marR genes of Shigella isolates and its association. MATERIALS AND METHODS: Fifty three isolates of Shigella spp. were tested to evaluate the antimicrobial susceptibility by disc diffusion method (Kirby-Bauer) according to the Clinical Laboratory Standard Institute (CLSI) for the following antimicrobials: ciprofloxacin, norfloxacin, ampicillin, tetracycline, chloramphenicol, trimethoprim, gentamicin and streptomycin and mutation on marAR genes by using polymerase chain reaction–Single strand conformation polymorphism analysis. RESULTS: Study revealed that there was significant association in between resistant to ciprofloxacin, norfloxacin and gentamicin with mutation in marA gene (87.5% vs 51.1%, P&lt;0.05; 87.5%Vs 51.1%, P&lt;0.05 and 90% vs 48.8%, P&lt;0.05, respectively). However, there was no significant association in between resistant to tetracycline, streptocycin and ampicillin. Similarly, it was noted that the association in between antimicrobial resistance with mutation in marR like ciprofloxacin (O% vs 57.8%, P&lt;0.05); norfloxacin (O% vs 57.8%, P&lt;0.05), chloramphenicol (O% vs 70.3%, P&lt;0.05); gentamicin (O% vs 60.5%, P&lt;0.05) and trimethoprim (42.6% vs 100%, P&lt;0.05), suggest that mutation in marR is protective factor for antimicrobial resistance. CONCLUSIONS: The study revealed that mutation in marR is preventive factors for antimicrobial resistance like ciprofloxacin, norfloxacin, chloramphenicol, gentamicin and trimethoprim. DOI: http://dx.doi.org/10.3126/ijim.v1i2.7406 Int J Infect Microbiol 2012;1(1):43-48

Shigella strains isolated in Japan between 1971 and 1979 were surveyed for drug resistance and distribution of R plasmids. Of 2,510 strains, 89.3% were resistant to either one or various combinations of four drugs, tetracycline, chloramphenicol, streptomycin, and sulfanilamide. About 66% of the Shigella isolates were quadruply resistant. The frequency of isolation of R plasmids from quadruply resistant Shigella strains was the highest when compared with other strains resistant to various combinations of the four drugs. The conjugal transferability of 204 quadruply resistant strains isolated between 1977 and 1979 was tested by various mixed-culture methods. Among the total strains examined, 70.6% carried transferable resistance when tested by the conventional broth culture method, 90.2% transferred their resistance when, in addition the replica-plating method was used and 97.5% could transfer their resistance when the membrane filter method was also used. Although the remaining five strains could not transfer their resistance by any of the mixed culture methods, the drug resistance of four of the five strains was mobilized by the concomitant presence of F-tet or T-kan plasmid. These results indicate that almost all of the quadruple resistance in Shigella isolates was mediated by plasmid.

The susceptibility patterns of 35 Shigella isolates (16 S. flexneri, 14 S. dysenteriae and 5 S. sonnei) to trimethoprim (Tp) and various antibiotics including amoxycillin, amoxycillin-clavulanic acid, nalidixic acid, ciprofloxacin, ceftazidime and ceftriaxone, were investigated. Twenty-two (62.8%) strains were resistant to Tp with a minimal inhibitory concentration (MIC50) value of 512 mg/L. Only six isolates were amoxycillin resistant, to which clavulanic acid restored sensitivity in all of them. None of the isolates were resistant either to extended spectrum cephalosporins or to quinolones. Resistance to Tp was transferred from 7 of the 22 isolates (31.8%) to the recipient Escherichia coli K12. Tp MIC values of the transconjugants were 512 mg/L. In no strain could amoxycillin resistance be transferred. Our results indicate that as the prevalance of transferable Tp resistance in Shigella isolates in Izmir is substantially high, alternative antimicrobial agents should be considered for empirical antibiotic therapy.

Molecular Evaluation of High Fluoroquinolone Resistant Genes in Endemic Cases of Shigellosis, Northeast Part of Karnataka, India

Azithromycin (AZT) has widely been used for the treatment of shigellosis in children. Recent studies showed a high rate of decreased susceptibility to azithromycin due to different mechanisms of resistance in Shigella isolates. Accordingly, the purpose of this study was to investigate the role of azithromycin resistance mechanisms of Shigella isolates in Iran during a two-year period. In this study, we investigated the mechanisms of resistance among Shigella spp. that were isolated from children with shigellosis. The minimum inhibitory concentration (MIC) of Shigella isolates to azithromycin was determined by the agar dilution method in the presence and absence of Phe-Arg-β-naphthylamide inhibitor. The presence of 12 macrolide resistance genes was investigated for all isolates by PCR for the first time in Tehran province in Iran. Among the 120 Shigella spp., only the mph(A) gene (49.2%) was detected and other macrolide resistance genes were absent. The phenotypic activity of efflux pump was observed in 1.9% of isolates which were associated with over expression of both omp(A) and omp(W) genes. The high prevalence of the mph(A) gene among DSA isolates may indicate that azithromycin resistance has evolved as a result of antimicrobial selection pressures and inappropriate use of azithromycin.

Plasmid-mediated quinolone resistance in Shigella isolates over a decade in India.

BackgroundThe drug resistance is expected to be the most important challenge in infection control in Iran, where there is no local report or standard drug resistance monitoring system. Therefore, this study aimed to investigate the aerobic and anaerobic bacterial profile of nosocomial infections and their antibiotic resistance in Ahvaz, southwest Iran.MethodologyThe gram-positive and gram-negative bacteria were identified on the basis of conventional culture and biochemical tests. The antibiotic resistance of the bacterial isolates against antibiotics was determined by the disk diffusion method.ResultsAmong total 1156 collected positive samples, E. coli and coagulase-negative staphylococci (CoNS) were the most frequent pathogenic gram negative bacteria (GNB) and gram positive bacteria (GPB) respectively. Drug susceptibility testing revealed that among GNB, P. aeruginosa was 100% resistant to amikacin, cefepime, ciprofloxacin and tetracycline. In the case of E. coli, the resistance rate was (98%) for trimethoprim sulfamethoxazole and cefepime. For GPB, S. aureus showed the highest resistance rates to amikacin (100%) and clindamycin (100%). In addition, CoNS strains showed a high level of resistance to doxycycline (100%), erythromycin (100%) and cefoxitin (97%). In Bacteroeides fragilis isolates, the highest resistance rate belonged to clindamycin (72%), and Clostridium perfringens strains showed high level of resistance to penicillin (46%).ConclusionThe results highlighted that there are distinct factors leading to antimicrobial resistance in Ahvaz, southwest Iran. The primary contributors to the resistance development, include poor surveillance of drug-resistant infections, poor quality of available antibiotics, clinical misuse, and the ease of access to antibiotics. Moreover, similar factors such as self-medication and the lack of regulation on medication imports play a role in antibiotic resistance in the region.

Background: Urinary stones are one of the major health problems worldwide and a significant part of referrals to physicians. Also, growing antibiotics resistance is an important concern. The purpose of this study was to evaluate the prevalence of urinary stones and bacterial strains with antibiotic resistance in women admitted to shiraz, southwest of Iran, from 2014 to 2019. Methods: In this period, all women admitted to the Zeinabieh Hospital were evaluated. The frequency of urinary stones and bacterial strains and antibiogram patterns of bacterial isolates were investigated from 2014 to 2019.Antibacterial susceptibility testing was done by disk diffusion method, fulfilling the standard criteria of “Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility”. Results: Out of 101 isolates, the identified gram-negative bacteria were E. coli (68.3%), Klebsiella (6.9%), Enterobacter (6.9%), Pseudomonas (5%), Proteus (1%), and Acinetobacter (1%). Of gram-positive strains, Coagulase-positive Staphylococci (5.9%), coagulase-negative Staphylococcus (CONS) (3%) and Non-Hemolytic Streptococci (2%) were identified. Also, the frequency of urinary tract stones was about 5%. Four cases of kidney stones were associated with E. coli and one case with Klebsiella. All isolates had the highest resistance to co-trimoxazole (62.5%), Ampicillin and Nalidixic acid.Conclusion: In this study, no association was found between the type of bacteria and urinary stone. From the variable bacteria isolated, the highest rate of E. coli (as an important pathogen) was confirmed. Also, most of the bacterial isolates were resistant to multiple antibiotics, although Amikacin and nitrofurantoin (as the empirical therapy in our hospital) were dominated.