Intra-hospital dissemination of clinical and environmental isolates of Stenotrophomonas maltophilia from Tehran.

Stenotrophomonas maltophilia isolates are responsible for various hospital-acquired infections and are particularly increasing in the immunocompromised patients. The aim of this study was to determine the clonal relatedness between S. maltophilia isolates originating from the clinic and environment. A total of 150 S. maltophilia isolates from patients and 1108 environmental samples obtained in three hospitals from Tehran. Following molecular identification targeting 23S rRNA gene, the clonal relatedness of the environmental and clinical isolates was determined using pulsed field gel electrophoresis (PFGE). Of the 150 clinical and 18 environmental isolates identified using phenotypic tests, the speciation of 120 and 15 was confirmed by targeting the 23S rRNA gene. The 24 common pulsotypes (PTs) and 32 single PTs were identified by PFGE. Only a small cluster was shared among the clinic and environment within a hospital; therefore, the intra-hospital dissemination of certain isolates of S. maltophilia among the clinic and environment was demonstrated.

Molecular typing of cytotoxin-producing Klebsiella oxytoca isolates by 16S-23S internal transcribed spacer PCR.

Cytotoxin is one of the important pathogenic factors, which plays a role in the virulence of Klebsiella oxytoca. The aim of this study was to investigate molecular typing of clinical isolates of the cytotoxin-producing K. oxytoca using internal transcribed spacer (ITS) PCR. A total of 75 isolates of K. oxytoca were isolated from clinical samples; they were verified as K. oxytoca by standard microbiological tests and PCR. Production of toxin determines the cytotoxic effects on HEp-2 cells. The genetic diversity of isolates of the cytotoxin-producing K. oxytoca were defined by ITS-PCR. Of all the isolates investigated, five K. oxytoca strains isolated from stool cultures, two strains from blood samples, one strain from a wound and one strain isolated from urine had cytotoxic effects on HEp-2 cells. The ITS-PCR patterns showed genetic diversity among cytotoxin-producing isolates. The ITS-PCR method had good discriminatory power; performance of this method and interpretation of the results were easy and repeatable. Five genetic diversity patterns were identified by ITS-PCR.

Pathogenicity locus determinants and toxinotyping of Clostridioides difficile isolates recovered from Iranian patients.

Little is known about the toxin profiles, toxinotypes and variations of toxin Clostridioides difficile C (tcdC) in Iranian C. difficile isolates. A total of 818 stool specimens were obtained from outpatients (n = 45) and hospitalized patients (n = 773) in Tehran, Iran, from 2011 to 2017. The 44 C. difficile isolates were subjected to PCR of toxin C. difficile A (tcdA), toxin C. difficile B (tcdB), tcdA 3'-end deletion, toxinotyping and sequencing of the tcdC gene. Thirty-eight isolates (86.36%) were identified as tcdA and tcdB positive, and the remaining six isolates (13.63%) were nontoxigenic. All tcdA- and tcdB-positive isolates yielded an amplicon of 2535 bp by PCR for the tcdA 3' end. Fourteen (36.84%), seventeen (44.73%) and seven (18.43%) isolates belonged to wild-type, toxin C. difficile C subclone3 (tcdC-sc3) and tcdC-A genotype of tcdC, respectively. Thirty-one isolates (81.57%) belonged to toxinotype 0, and seven isolates (18.42%) were classified as toxinotype V. This study provides evidence for the circulation of historical and hypervirulent isolates in the healthcare and community settings. Furthermore, it was also demonstrated that the tcdC-A genotype and toxinotype V are not uncommon among Iranian C. difficile isolates.

Highly antibiotic-resistant Clostridium difficile isolates from Iranian patients.

AIMS: Little is known about the resistance rate and susceptibility profile of Clostridium difficile isolates in Iran. Therefore, the aim of present study is to assess the rate of drug-resistant C. difficile. METHODS AND RESULTS: During a 6-year period, four hospitals submitted 735 stool specimens from patients suspected for C. difficile infections to the anaerobic bacteriology laboratory. The 46 C. difficile isolates were subjected to disc diffusion and minimum inhibitory concentration (MIC) Test Strips. All isolates were susceptible to vancomycin (VAN) while the highly resistant phenotypes of metronidazole (MTZ) (67·4%), moxifloxacin (78·3%), ciprofloxacin (69·5%) and tetracycline (82·6%) were observed. Of more concern, 67·3% of C. difficile isolates displayed multidrug-resistant phenotypes. More than half of the isolates (n = 27, 58·6%) were coresistant to ciprofloxacin and moxifloxacin. The MIC90 of VAN was ≤2 mg l-1 , whereas this value for MTZ, ciprofloxacin, moxifloxacin and tetracycline was higher than the resistance breakpoints. According to the comparison of interpretive categories for two tests, the categorical agreement was less than 90% for VAN, ciprofloxacin and tetracycline. CONCLUSIONS: The disc diffusion method can be used to detect the isolates with reduced susceptibility to MTZ or moxifloxacin. The high rate of resistance to fluoroquinolones highlights the possibility of the emergence of hypervirulent strains in our settings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data regarding the high level of resistance against multiple antibiotics except VAN.

The impact of primer sets on detection of the gene encoding biofilm-associated protein (Bap) in Acinetobacter baumannii: in silico and in vitro analysis.

The Acinetobacter baumannii virulence protein Bap is encoded by a large gene and contains both variable sequence and repetitive modules. To date, four primer sets targeting different regions of bap have been designed, but no study has evaluated all these primers simultaneously for detection of bap. Here, we assessed the effect of primer sets Bap I-IV, on detection of bap both in silico and in vitro. Using the primer set Bap II, all 143 tested strains yielded an amplicon corresponding to the bap gene. This primer set showed the highest sensitivity (100, 95% CI: 97·9-100%) compared to the other primer sets. This study demonstrates that primer set Bap II performs with optimal efficiency for detection of the bap gene among different strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study investigated the effect of nucleotide variation on PCR detection of the bap gene in various Acinetobacter baumannii strains. Since bap is the target gene for many detection assays, this variation can affect the detection efficiency. Here we present a primer set Bap II with optimal detection efficiency amongst 143 different strains, as shown by in silico and in vitro evidence.

Inhibitory effect of Lactobacillus plantarum and Lb. fermentum isolated from the faeces of healthy infants against nonfermentative bacteria causing nosocomial infections.

Nosocomial infection constitutes a major public health problem worldwide. Increasing antibiotic resistance of pathogens associated with nosocomial infections has also become a major therapeutic challenge for physicians. Thus, development of alternative treatment protocols, such as the use of probiotics, matters. The aim of this research was to determine the antagonistic properties of Lactobacillus plantarum and Lb. fermentum isolated from the faeces of healthy infants against nonfermentative bacteria causing nosocomial infections. One hundred five samples of nosocomial infections were collected and processed for bacterial isolation and antimicrobial susceptibility testing following standard bacteriologic techniques. The antibiotic susceptibility test was performed by the disk diffusion method, and antagonistic effect of Lactobacillus strains was investigated by well diffusion method. Of 105 samples, a total of 29 bacterial strains were identified as nonfermentative bacteria, including 17 Acinetobacter baumannii and 12 Pseudomonas aeruginosa. A. baumannii showed high resistance to tested antibiotics except ampicillin/sulbactam, and P. aeruginosa showed resistance to ampicillin/sulbactam and gentamicin and sensitive to amikacin and meropenem. Lb. plantarum had antagonistic properties against both A. baumannii and P. aeruginosa strains. Lb. plantarum had considerable effects on preventing the growth of A. baumannii and P. aeruginosa strains. However, further research is needed to better understanding of these effects on P. aeruginosa.

Trend of extensively drug-resistant Acinetobacter baumannii and the remaining therapeutic options: a multicenter study in Tehran, Iran over a 3-year period.

Comprehensive data on drug-resistant patterns of Acinetobacter baumannii isolates in developing countries is limited. We conducted a multihospital study to assess the rate and trend of drug-resistant phenotypes in Ac. baumannii using standardized definitions and to determine the remaining therapeutic options against resistant phenotypes. The 401 nonduplicate isolates were collected from six hospitals which are geographically distributed across Tehran, Iran over a 3-year period. Following PCR of blaOXA-51-like gene, susceptibility testing was performed against nine antimicrobial agent categories. Three hundred and ninety (97%) isolates were resistant to least two carbapenems; carbapenem-resistant Ac. baumannii. The majority of isolates (366, 91·3%) were extensively drug resistant (XDR) and the rest of the isolates were classified as multidrug resistant (26, 6·8%) and susceptible (9, 2·2%). The rate of XDR-AB slightly decreased from 93·8% in 2011 to 89·8% in 2013. A considerable decrease in resistance to doxycycline, minocycline and tigecycline was demonstrated. The XDR-AB isolates showed susceptibility to gentamicin (10·4%), tobramycin (23%), ampicilin-sulbactam (30·1%), minocycline (32·8%), tigecycline (10·7%), doxycycline (21·6%), colistin (100%) and polymixin B (100%). We demonstrated the rising trend of resistance to all antibiotic categories except tetracyclines and folate pathway inhibitors. We found that the treatment options against XDR-AB are extremely limited and each treatment alternative including even old, but safe, antibiotics might be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: The high frequency of drug-resistant phenotypes including carbapenem-resistant Acinetobacter baumannii, multidrug-resistant, and extensively resistant has been demonstrated in Ac. baumannii isolates tested here. As the antibiotic resistance pattern of isolates varies in different geographical regions, this study can provide comprehensive information about the antibiotic resistance profile of Ac. baumannii isolates in Tehran. In addition, the resistance profiles could be effectively considered by clinicians to manage antibiotic therapy. This work also emphasizes on the prudent use of antibiotics and the monitoring of antibiotic susceptibility trend and rate.

Comparative in vitro activity of carbapenems against clinical isolates of Acinetobacter baumannii.

AIMS: The aim of this multi-hospital study was to assess the in vitro activity of doripenem and its comparators, imipenem and meropenem, using the new CLSI breakpoints against a large population of a frequently isolated nosocomial pathogen, Acinetobacter baumannii. METHODS AND RESULTS: During a 2-year period, four referral or tertiary hospitals submitted 400 isolates of Ac. baumannii for susceptibility testing using imipenem, meropenem and doripenem via disc diffusion and E-test methods. A subset of 390 isolates was resistant to all three tested carbapenems. Doripenem and meropenem (MIC50 , 32 µg ml(-1) ) had comparable activity, albeit doripenem's activity was greater than imipenem (MIC50 , >32 µg ml(-1) ). A significantly higher proportion of the isolates were inhibited by doripenem than by imipenem at MIC values of 12, 16, 24 and 32 µg ml(-1) (P < 0·05). The cumulative percentage of imipenem MICs was lower compared to its comparators. The comparison of resistance rate to imipenem and meropenem based on old and new breakpoints showed <1% difference. The overall agreement between the two susceptibility testing methods was ≥95%. CONCLUSION: Doripenem has a slightly greater in vitro activity than imipenem in terms of zone breakpoints and MIC values, but its activity is comparable to meropenem. SIGNIFICANCE AND IMPACT OF THE STUDY: Doripenem should be considered as a therapeutic option for monotherapy or combination therapy, particularly when the therapeutic options are limited.

Heterogeneity in resistant fecal Bacteroides fragilis group collected from healthy people.

Normal nonpathogenic flora would represent a constant lake of resistance genes potentially transferable to human pathogens. To assess the prevalence of resistance genes and genetic variability of Bacteroides fragilis group (BFG) from normal flora, 177 Bacteroides isolates obtained from the fecal samples of healthy individuals. These isolates were subjected to antibiotic susceptibility testing and pulsed field gel electrophoresis (PFGE). The isolates were further tested for the presence of ermF, tetQ and bft genes by PCR. Our results indicated the presence of different clonal strains (1 common type and 57 single types) among the resistant isolates. The resistance rate for the six antibiotics in this study was between 1% and 95%. Most of the isolates (99%) were susceptible to metronidazole. ermF and tetQ were detected in all erythromycin and tetracycline resistant isolates. None of the isolates were carried bft gene. These data suggest dissemination of heterogenic clonal groups in healthy persons and resistance to 5 high commonly used antibiotics.

Molecular analysis of pbp2b in Streptococcus pneumonia isolated from clinical and normal flora samples.

Streptococcus pneumoniae is an important bacterial pathogen responsible for respiratory infections, bacteraemia, and meningitis remains an important cause of disease and mortality in infants and younger children around the world, with penicillin being considered the drug of choice for the treatment of infections. However, penicillin-resistant S. pneumonia is now becoming endemic worldwide. In this study, a total of 80 pneumococcal isolates were collected from different clinical sources as well as normal flora. These isolates were subjected to antimicrobial susceptibility testing and MIC determination. The penicillin-binding proteins, pbp2b, were amplified by PCR, and they were sequenced. The genetic relationship of the penicillin-resistant isolates was performed by BOX PCR. Overall, 36 pneumococcal (45 %) isolates were found to be resistant to penicillin with different MICs. The majority of them (80 %) were intermediately resistant with MIC of 0.12-1 µg/ml, whereas 20 % of isolates were penicillin resistant with MICs of >2 µg/ml. The results identified seven groups which were based on the amino acid substitutions of pbp2b. Sequencing analysis revealed that the most prevalent mutation was the substitution of Adenine for Thymine at the position 445 which is next to the second PBP2b-conserved motif (SSN). This study indicates that resistance to penicillin appears to be dependent on specific mutations in pbp2b, and the substitution in S620 → T near to the third PBP2b-conserved motif appears to be important in developing highly antibiotic-resistant isolates. Moreover, there was a positive correlation between the mutations in pbp2b gene and MIC.

Isospora belli associated recurrent diarrhea in a child with AIDS.

Persistent diarrhea is a major manifestation of Acquired Immunodeficiency Syndrome (AIDS) which might be more complicated in Human Immunodeficiency Virus (HIV) infected children especially those from developing countries. There are numerous reports showing the emergence of intestinal opportunistic coccidian parasites, mostly Cryptosporidium parvum and Isospora belli in HIV-infected individuals. The prevalence of isosporiasis is probably underestimated in developing countries because routinely not all HIV-infected patients are examined for the presence of this protozoan infection. Here we report a case of HIV-infected isosporiasis presenting with failure to thrive and persistent diarrhea. Since I. belli infection in children responds well to therapy with trimethoprim-sulfamethoxazole, isosporiasis should be considered as a treatable infection in AIDS, if it is detected at proper time.

Molecular identification of Pseudomonas aeruginosa recovered from cystic fibrosis patients.

OBJECTIVE: Precise identification of various morphotypes of Pseduomonas aeruginosa which developed during cystic fibrosis (CF) is of prime importance. We aimed to identify the isolates of P. aeruginosa recovered from CF patients at the genus and species level through primers targeting oprI and oprL genes via PCR. METHODS: Sputum samples or throat swabs were taken from 100 CF patients and plated on cetrimide agar. All suspected colonies were primarily screened for P. aeruginosa by a combination of phenotypic tests. Molecular identification of colonies was performed using specific primers for oprI and oprL genes. RESULTS: Based on phenotypic tests, P. aeruginosa isolates were recovered from 40% of CF patients. Forty isolates yielded amplicon of oprI gene using genus-specific primers confirming the identity of fluorescent pseudomonads. However, 37 of 40 isolates yielded amplicon of oprL gene using species-specific primers, verifying the identity of P. aeruginosa. CONCLUSION: This study showed that the species-specific PCR targeting oprL gene can be used as accurate test for identification of highly adaptable P. aeruginosa in CF patients. This procedure may provide a simple and reliable method for identification of various morphotypes.