Collateral sensitivity between aminoglycosides and beta-lactam antibiotics depends on active proton pumps.

BACKGROUND: Selection inversion is the hypothesis for antibiotic resistant inhabitation in bacteria and collateral sensitivity is one of the proposed phenomena for achievement of this hypothesis. The presence of collateral sensitivity associated with the proton motivation pump between the aminoglycosides and beta-lactam group of antibiotics is one of the examples of collateral sensitivity in some studies. The aim of this study was to demonstrate that collateral sensitivity between aminoglycosides and beta-lactam antibiotics associated with proton motivation pump may not be true in all cases. METHODS: In this study, 100 Pseudomonas aeruginosa were surveyed. Gentamicin and imipenem-resistant strains were confirmed by disc diffusion method and MIC. Active proton motivation pumps were screened by pumps inhibitor. Semi-quantitative Real-Time PCR assay was used to confirm gene overexpression. RESULTS: Seventy-six and 79 out of 100 strains were resistant to gentamicin and imipenem, respectively. Seventy-five strains were resistant to both gentamicin and imipenem. The results of proton pump inhibitor test showed the involvement of active proton motivation pump in 22 of 75 imipenem- and gentamicin-resistant strains. According to Real - Time PCR assay, mexX efflux gene was overexpressed in the majority of isolates tested. DISCUSSION: The collateral sensitivity effect cannot explain the involvement of active proton motivation pumps in both imipenem and gentamicin-resistant strains simultaneously. Active and/or inactive proton pump in gentamicin-sensitive and/or resistant strains cannot be a suitable example for explanation of collateral sensitivity between aminoglycosides and beta-lactam antibiotics.

Investigation of Efflux-Mediated Tetracycline Resistance in Shigella Isolates Using the Inhibitor and Real Time Polymerase Chain Reaction Method.

BACKGROUND: Shigella spp. are gram negative bacteria, which are of global public health importance. The growing of multidrug-resistant Shigella isolates are a major problem around the world. METHODS: Overall, 50 isolates of Shigella spp. from children diarrheic stools were studied. The isolates were identified and confirmed using biochemical, serological and molecular methods (ipaH, wbgZ and rfc genes). Antimicrobial susceptibility test was done according to the Clinical and Laboratory Standards Institute (CLSI) guidelines against minocycline, tetracycline, doxycycline, ampicillin, streptomycin, trimethoprim-sulfamethoxazole, nalidixic acid, norfloxacin, ciprofloxacin and levofloxacin. Also, the role of efflux pump in defense of Shigella against tetracycline was investigated by Minimum Inhibitory Concentration (MIC) with and without an efflux pump inhibitor. Detection of tetA, tetB, tetC and tetD genes in Shigella was evaluated by conventional Polymerase Chain Reaction (PCR) and real time PCR. RESULTS: Molecular identification revealed a prevalence of 14% for Shigella flexneri and 86% for Shigella sonnei. Minimum Inhibitory Concentration (MIC) of 90% of resistant isolates was changed in the presence CCCP. Results of conventional PCR exhibited that 66% of isolates were positive for tetA, while according to real time PCR method, 90% of isolates carried tetA. Positive results for tetB were 12% and 18% by conventional and real time PCR methods, respectively. No positive results were detected for tetC and tetD. Also, tetB was detected only in S. flexneri while tetA was detected in both S. flexneri and S. sonnei. CONCLUSION: It seems that efflux-mediated tetracycline resistance to tetracycline in S. flexneri can be related to tetB, however resistance in S. sonnei can be related to the expression of tetA.

Multi-locus sequence type analysis of Shigellas pp. isolates from Tehran, Iran.

BACKGROUND AND OBJECTIVES: Strains of Shigella spp. can cause shigellosis, or bacillary dysentery. that is a public health problem worldwide. The aim of this study was to describe the population structure and genetic relatedness of multidrug resistant S. sonnei and S. flexneri isolated during a one year period from children with diarrhea in Tehran, Iran. MATERIALS AND METHODS: A total of 70 Shigella spp. were detected during the study period. Twenty MDR isolates of Shigella spp. were randomly selected and used in this study. Bacterial identification was performed by conventional biochemical and serological and confirmed by molecular method. After antimicrobial susceptibility testing, we used Multilocus sequence typing (MLST) for subtyping isolates. RESULTS: We found 14 Shigella sonnei and 6 Shigella flexneri isolates. Results of MLST showed five sequence types (ST) (145, 152, 241, 245, 1502) and BURST analysis revealed the largest number of single locus variant (SLV) and highest frequency (FREQ) for ST152. ST 152 with nine members was predicted as the founder by BURST. Frequency for ST 1502 and ST 245 was four isolates and the least frequency was seen for ST 241 and 145 with one and two members, respectively. ST 145 and ST 245 were described as singletons in BURST. All isolates with ST145 and ST245 were identified as Shigella flexneri. CONCLUSION: Annual Multi locus sequence typing of MDR Shigella would help us in better understanding of dominant species and comparing our results with the same studies in other countries especially our neighbor countries in source tracking purposes.

Analysis of antibiotic consumption in burn patients.

Infection control is very important in burn care units, because burn wound infection is one of the main causes of morbidity and mortality among burn patients. Thus, the appropriate prescription of antibiotics can be helpful, but unreasonable prescription can have detrimental consequences, including greater expenses to patients and community alike. The aim of this study was to determine the effect of antibiotic therapy on the emergence of antibiotic-resistant bacteria. 525 strains of Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus were isolated from 335 hospitalized burn patients. Antibiotic susceptibility tests were performed after identification the strains. The records of patients were audited to find the antibiotic used. The results indicated that P. aeruginosa is the most prevalent Gram-negative bacteria. Further, it showed a relation between abuse of antibiotics and emergence of antibiotic resistance. Control of resistance to antibiotics by appropriate prescription practices not only facilitates prevention of infection caused by multi-drug resistant (MDR) microorganisms, but it can also decrease the cost of treatment.

Acinetobacter baumannii clonal lineages I and II harboring different carbapenem-hydrolyzing-ß-lactamase genes are widespread among hospitalized burn patients in Tehran.

The aim of this study was to analyze antimicrobial resistance patterns and their encoding genes and genotypic diversity of Acinetobacter baumannii isolated from burn patients in Tehran, Iran. The presence of extended-spectrum beta-lactamase- and blaOXA-encoding genes among 37 multidrug resistant (MDR) A. baumannii strains isolated from patients hospitalized in a teaching hospital in Tehran was evaluated. Susceptibility to 7 antibiotics was tested by disk agar diffusion and to polymyxin B and colistin was tested by E-test, according to CLSI guidelines. All isolates were then analyzed by PCR for the presence of blaIMP, blaVIM, blaSIMblaOXA-23, blaOXA-24, and blaOXA-58-like carbapenemase genes, and blaOXA-51-like, blaTEM, blaSHV, blaPER, blaVEB, and blaGIM genes. Genotyping of A. baumannii strains was performed by repetitive sequence-based (REP)-PCR and cluster analysis of REP-PCR profiles. A. baumannii isolates were assigned to international clones by multiplex PCR sequence group analysis. Twenty-five A. baumannii isolates were classified as MDR, and 12 were classified as extensively drug resistant. All isolates were susceptible to colistin and polymyxin B. Eighty-one percent of the isolates was resistant to imipenem or meropenem and harbored at least one or both of the blaOXA-23-like or blaOXA-24-like carbapenemase genes. Co-existence of different resistance genes was found among carbapenem-resistant isolates. Multiplex PCR sequence group analysis most commonly assigned A. baumannii isolates to international clones I (18/37; 48.6%) and II (18/37; 48.6%). An alarming increase in resistance to carbapenems and the spread of blaOXA-23-like and/or blaOXA-24-like carbapenemase genes was observed among A. baumannii strains belonging to clonal lineages I and II, isolated from burn patients in Tehran.

Characterization of Carbapenemases in Extensively Drug Resistance Acinetobacter baumannii in a Burn Care Center in Iran.

The emergence of multidrug resistance (MDR) and extensively drug resistance (XDR) in Acinetobacter baumannii has made an important challenge in the treatment of infections caused by this organism. The ability of carbapenemase production is one of the main mechanisms for the emergence of MDR and/or XDR in A. baumannii. The aim of this study was to detect carbapenemase producer A. baumannii. In this study, 65 imipenem resistant A. baumannii were collected from burned patients. Biochemical identification, antibiotic susceptibility test and multiplex polymerase chain reactions for the detection of carbapenemases genes were performed. The results showed that all strains carried bla OXA-51. 83%, 12.5% and 9.23% strains harbored bla OXA-23, bla VIM and bla KPC genes, respectively. None of the isolates carried bla IMP, bla OXA-48, blaNDM-1 and bla SPM-1 genes. The results of this study indicate the emergence of Klebsiella pneumoniae Carbapenemase (KPC) in A. baumannii causing nosocomial infections in burned patients which can be important for hospital infection prevention systems in Iran.

Effect of Efflux Pump Inhibitor Carbonyl Cyanide 3-Chlorophenylhydrazone on the Minimum Inhibitory Concentration of Ciprofloxacin in Acinetobacter baumannii Clinical Isolates.

BACKGROUND: Acinetobacter baumannii is an important human pathogen with increasing notoriety in the recent years, as a causative organism of drug resistant nosocomial infections, particularly in immunocompromised patients hospitalized in burn centers. OBJECTIVES: The aim of this study was to determinate the role of efflux pump(s) in ciprofloxacin resistance of A. baumannii strains isolated from burn patients. MATERIALS AND METHODS: Sixty-five A. baumannii strains were isolated from the burn patients hospitalized in Motahari Burns and Reconstruction Center in Tehran, Iran. Susceptibility test to ciprofloxacin was carried out by disk agar diffusion and agar dilution methods, according to the CLSI guidelines. Activity of the efflux system was evaluated using efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). RESULTS: All Acinetobacter isolates were resistant to ciprofloxacin. The Minimum inhibitory concentration (MIC) range of ciprofloxacin in isolates was 4 to 128 µg/mL or greater. Moreover, susceptibility of strains to ciprofloxacin was highly increased in the presence of efflux pump inhibitor; So that, for 86.1% (56/65) of isolates, CCCP reduced the MIC by 2 to 64 folds. CONCLUSIONS: Our findings are suggestive that efflux-based system may play a role in fluoroquinolone resistance in A. baumannii isolates, affecting hospitalized patients. The ability of Acinetobacter to acquire resistance to these potent antimicrobials by the efflux pump mechanism is a concern. Therefore, new strategies are required in order to eliminate the efflux transport activity from the resistant bacteria causing nosocomial infections and provide more appropriate approaches for treatment and management of troubling infections.

Characterisation of SCCmec elements in methicillin-resistant Staphylococcus aureus isolated from burn patients.

Staphylococcus aureus is an important pathogen, especially in burn units all around the world. Because of the emergence of the ß-lactam antibiotic-resistant strains since 1961, concern about the prevalence of methicillin-resistant S. aureus (MRSA) has increased in these units. Resistance to methicillin is mediated by penicillin-binding proteins (PBPs) that have enough affinity for binding to the ß-lactam ring, but another kind of protein (PBP2α), which is encoded by the mecA gene, has a lower affinity for binding to these antibiotics. The mecA gene is transferred by SCCmec (staphylococcal cassette chromosome mec) as a mobile genetic element, exclusively found in the Staphylococcus genus. Identification of the frequency of the mecA gene, different SCCmec types and also its incidence may have benefit in surveillance prevention and control of MRSA strains in burn units. In this study, 40 S. aureus isolates were collected from patients hospitalised in Motahari burn center of Tehran, during 2012-2013. Conventional microbiological methods were applied and the confirmed isolates were stored at -20°C for molecular polymerase chain reaction (PCR) tests. The antibiotic resistance pattern was performed by disc diffusion method and finally the different SCCmec types were determined by specific primers. During this research, 40 isolates of S. aureus were collected from burn patients, of which (37.5%) of the specimens belonged to female patients and 62.5% to male patients. The aetiology of the burn was classified as follows: open flame (35%), liquid (32.5%), chemical (5%) and other (27.5%). By a disc diffusion method, no resistance pattern was observed to vancomycin and fosfomycin. Based on a multiplex PCR assay, the five different SCCmec types were detected as: 47.5% type III, 25% type IV, 10% type V, 10% type II and 7.5% type I.

Clonal dissemination of methicillin-resistant Staphylococcus aureus in patients and the hospital environment.

BACKGROUND: Methicillin is the drug of choice to treat infections caused by resistant strains of Staphylococcus aureus. However, methicillin-resistant S. aureus (MRSA) is now becoming endemic in many hospitals worldwide and is the cause of nosocomial outbreaks. METHODS: To assess clonality and dissemination of MRSA strains in the hospitals of Tehran, a total of 60 MRSA strains were isolated from hospitalized patients (n=44) and hospital equipment and environment (n=16) of three metropolitan hospitals in Tehran between July 2009 and March 2010. These strains were subjected to antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and biochemical fingerprinting using the PhPlate system. RESULTS: Results showed the presence of between one and three dominant clonal groups within each hospital, with most equipment and environmental strains being identical to the dominant clones of hospitalized patient strains. The rate of resistance of these strains to the 13 antibiotics tested ranging from 2% to 100%, with resistance being highest for penicillin, ciprofloxacin, and tetracycline (>98% of the isolates). Comparison of the strains isolated from the three hospitals using a combination of PFGE and PhP types showed the presence of 11 clonal groups of MRSA among these hospitals; of these, three common clonal groups also had identical antibiotic resistance patterns and were found in more than one hospital. CONCLUSIONS: These data suggest dissemination of a few dominant clonal groups of MRSA strains in hospitals in Tehran, with high level resistance to other commonly used antibiotics.

The immunohistochemistry and toluidine blue roles for Helicobacter pylori detection in patients with gastritis.

BACKGROUND: Helicobacter pylori, which is associated with many upper gastrointestinal diseases, is found in half of the population of the world. Several special stains and immunohistochemistry stain for H. pylori are available. The need for and usefulness of immunohistochemical (IHC) technique has been debated for years. Toluidine blue is a simple stain for microbiological studies and is easily available in laboratories. Therefore, this study was conducted to compare hematoxylin and eosin (H&E), Giemsa and toluidine blue staining with immunehistochemistry for detection of H. pylori in patients with gastritis and also to correlate the results of these staining methods with pathological grading. METHODS: We reviewed 54 consecutive gastric biopsy specimens stained by H&E and Giemsa as well as by toluidine blue and immunohistochemistry stains for H. pylori. RESULTS: H. pylori was positively identified by IHC in 43 (79.63%) patients, while positive samples were found in 18 (33.33%), 24 (44.44%) and 33 (61.11%) patients using H&E, Giemsa and toluidine blue staining methods. Our results showed that classical histological staining methods are not sensitive enough to identify low numbers or coccoid forms of organism, while toluidine blue and immunohistochemistry play an important role in detection of H. pylori infection. CONCLUSION: Toluidine blue has been proved to be much more reliable than H&E and Giemsa in detection of H. pylori. In addition, in post treatment biopsies and in biopsies with unexplained chronic active gastritis without histological evidence of H. pylori should have immunohistochemistry done to detect possible low density or coccoid form of organisms.

Phenotypic detection of Klebsiella pneumoniae carbapenemase among burns patients: first report from Iran.

BACKGROUND: Resistance to antimicrobial agents such as carbapenems among enterobacteriacea has been increasing, especially in Klebsiella pneumonia that produces variety of enzymes including Klebsiella pneumoniae carbapenemase (KPC). This study is the first report of its kind investigating the resistance to carbapenems among burns patients in Iran. METHOD: During a 6-month period, 28 hospitalized burn patients who required to be placed on broad spectrum antibiotics were studied. Isolated species identified by routine biochemical test. Susceptibility testing for these species was performed by recommended the CLSI guidelines method. The tested antibiotics included cefotaxime, cefepime, aztreonam, imipenem, amoxicillin+clavulonic acid, gentamicin, amikacin, tobramycin, tetracycline, and trimethoprim-sulfamethoxazole, and chloramphenicol. For determination of KPC in phenotypical forms, Modified Hodge Test was utilized as per CLSI recommendation. RESULTS: Thirty-five Klebsiella spp. were isolated from 28 hospitalized patients. Nineteen out of 35 Klebsiella isolates were resistant to imipenem and that all of them had positive KPC. Nine of imipenem resistant isolates were also resistant to all tested antibiotics. Mortality rate among patients with positive KPC was 33%. CONCLUSION: High rate of multi-drug resistant (MDR) strains in isolates with positive KPC is a major challenge in Iran and that it could cause an increase in both mortality and morbidity among burn patients. Thus, appropriate infection control measures and guidelines are needed to prevent such infections among burn patients.

Epidemiological study of self-inflicted burns in Tehran, Iran.

Self-inflicted burns have been considered a serious mental health problem throughout the world and especially in economically developing countries. The aim of this study was to identify the epidemiologic features and current etiological factors of suicide by burning in Tehran, Iran, to develop effective prevention programs. Over a period of 2 years from March 1997 to March 1999, of the 1,236 patients aged 14 years and older who were admitted to Tohid Burn Center in Tehran, Iran, 110 (8.9%) had attempted suicide by self-immolation. The median age was 25 years (range: 14-68 years) and the median extent of the burns was 71% TBSA (range: 20-100%). Forty-eight patients had a previous psychiatric diagnosis (43.6%). Depression was the most common psychiatric diagnosis by history. The method most commonly used was a flame with the addition of a flammable liquid (frequently kerosene). Also, most of the patients (70%) were of a low socioeconomic class. Overall, self-inflicted burns should be considered an increasing mental health problem in our society. Therefore, it is necessary to implement prevention programs and strategies to reduce the incidence rate of this problem.