Bromhexine and its inhibitory effect on lipase - kinetics and structural study.

Lipase hydrolyses the ester bonds in triglyceride. It is an important enzyme in medicine and industry. Some pathogen bacteria use this exoenzyme to disrupt the extracellular matrix of host organisms. Pseudomonas uses various extracellular enzymes such as lipase to invade its host. In this report, for the first time, bromhexine was introduced as an inhibitor of lipase. Bromhexine is a mucolytic drug which is used in the treatment of respiratory tract disorders. The results showed that bromhexine inhibited the enzyme by competitive inhibition. IC50 and Ki values of the drug were 0.049 mM and 0.02 mM, respectively. Arrhenius plot showed that the drug reduced the activation energy. The enzyme was purified and SDS-PAGE showed that its molecular weight is 13 kDa. Fluorescence measurement revealed that binding of the drug to lipase could make structural changes in the enzyme. Inhibition of lipase by bromhexine could be applicable in medicine.

Evaluation of Outer Membrane Vesicles Obtained from Predominant Local Isolate of Boredetella pertussis as a Vaccine Candidate.

Background: Pertussis is a current contagious bacterial disease caused by Bordetella pertussis (Bp). Given the prevalence of pertussis, development of new vaccines is important. This study was attempted to evaluate the expression of main virulence factors (pertussis toxin [PTX], PRN [pertactin], and filamentous hemagglutinin [FHA]) from Bp predominant strains and also compare the expression of these factors in the outer membrane vesicles (OMVs) obtained from predominant circulating Bp isolate. Methods: The physicochemical features of the prepared OMVs were analyzed by electron microscopy and SDS-PAGE. The presence of the mentioned virulence factors was confirmed by Western blotting. BALB/c mice (n = 21) immunized with characterized OMVs were challenged intranasally with sublethal doses of Bp, to examine their protective capacity. Results: Electron microscopic examination of the OMVs indicated vesicles within the range of 40 to 200 nm. SDS-PAGE and Western blotting demonstrated the expression of all three main protective immunogens (PTX, PRN, and FHA), prevalent in the predominant, challenge, and vaccine strains, and OMVs of the predominant IR37 strain and BP134 vaccine strain. Significant differences were observed in lung bacterial counts between the immunized mice with OMV (30 CFU/lung) compared to the negative control group ((6 104 CFU/lung; p < 0.001). In mice immunized with OMVs (3 µg), the number of lungs recovered colonies after five days dropped at least five orders of magnitude compared to the control group. Conclusion: OMVs obtained from circulating isolates with the predominant profile may constitute a highly promising vaccine quality. They also can be proposed as a potential basic material for the development of new pertussis vaccine candidate.

Potential of a Bacteriophage Isolated from Wastewater in Treatment of Lobar Pneumonia Infection Induced by Klebsiella pneumoniae in Mice.

The potential of bacteriophages as alternative treatment for multidrug-resistant (MDR) Klebsiella pneumoniae-related infections has recently gained much interest. The purpose of this research was to isolate and characterize a K. pneumoniae-specific lytic phage with the potential to treat experimental lobar pneumonia induced by K. pneumoniae in mice. A lytic phage was isolated from an urban wastewater sample in Tehran and characterized by transmission electron microscopy (TEM), thermal, pH, and chloroform stability before being employed for treatment of mice infected with K. pneumoniae in an experimental model of lobar pneumonia. BALB/C mice were challenged by intranasal inoculation with 108 colony-forming units (CFU/ml) of K. pneumoniae ATCC 10031 followed by an intraperitoneal injection of the isolated phage using 1010 and 109 plaque-forming units (PFU/ml) simultaneously or 24 h post infection. Control groups of mice received bacteria or bacteriophage alone. Mice were euthanized daily up to 7 days post infection and examined for abnormality in their lungs and livers followed by determining the number of phages and bacteria in plasma and lung homogenates. The isolated phage (vB_KpnM-Teh.1) belonged to the Myoviridae family, was stable at 37 °C, pH 7, and was resistant to chloroform. Treatment of mice with a single dose of phage simultaneously at the time of infection, or 24 h post infection, resulted in seven and five logs decrease of CFU/ml in the lung homogenates up to 3 days after phage administration, respectively. The isolated phage may have the potential as a therapeutic agent against K. pneumoniae infections.

Prevalence of Extended-Spectrum ß-lactamase and Integron Gene Carriage in Multidrug-Resistant Klebsiella Species Isolated from Outpatients in Yazd, Iran.

BACKGROUND: Community-acquired infections by multidrug-resistant (MDR), extended-spectrum ß-lactamase (ESBL) producing Klebsiella species (Klebsiella spp.) is of major concern worldwide. Antibiotic resistance, production of extended-spectrum ß-lactamases (ESBLs), and carbapenemases, as well as the presence of classes 1, 2, and 3 integrons in outpatient isolates of Klebsiella collected from Yazd central laboratory, Yazd, Iran. METHODS: We collected 250 Klebsiella isolates from Yazd central laboratory between August 2015 and October 2017. Antibiotic susceptibility was determined against 18 antibiotics by disc diffusion, and multidrug-resistant isolates were tested for ESBL production by the phenotypic confirmatory test according to CLSI 2017 protocols. The amplification of ß-lactamase genes blaSHV , blaTEM , blaCTX-M , blaOXA-48 , blaKPC , and blaNDM , classes 1, 2, and 3 integrase genes, was carried out using specific primers and polymerase chain reaction (PCR). RESULTS: Of the 250 Klebsiella outpatient isolates, 3.6% were K. oxytoca and the rest were K. pneumoniae. Disc diffusion showed that 21 (8.4%) isolates were MDR, 19 (90.4%) of which were ESBL producers including one K. oxytoca. The most prevalent ß-lactamase gene was blaSHV followed by blaTEM and blaCTX-M , but blaOXA-48 , blaKPC , and blaNDM were not detected. Class 1 integron was detected in 18 out of 21 MDR isolates (85.7%), but classes 2 and 3 were not observed. Two isolates were resistant to carbapenems and harbored blaSHV , blaTEM , and blaCTX-M , as well as class 1 integron. CONCLUSION: ESBL production and the presence of multiple ß-lactamase genes in MDR community isolates of Klebsiella spp. can have significant implications in terms of the spread of these opportunistic pathogens.

Localization of Staphylococcus inside the vacuole of Candida albicans by immunodetection and FISH.

In our previous study, two bacteria B1 and B2 were excised from two amphotericin B-treated Candida albicans Y1 and Y2, respectively. Bacteria were identified as B1: Staphylococcus hominis and B2: Staphylococcus haemolyticus according to their biochemical characteristics and detection and sequencing of Staphylococcus-specific genes. In this study the intracellular origin of staphylococci inside the vacuole of yeast was examined. Polyclonal antibodies against S. hominis and S. haemolyticus were raised in rabbit and used for detection of staphylococcal proteins in protein pool of yeasts by western blotting (WB). Fluorescein-isothiocyanate (FITC)-conjugated antibodies were used for bacterial localization inside yeast's vacuole by direct immunofluorescence (DIF). Fluorescent in situ hybridization (FISH) with Staphylococcaceae -specific probe was performed for validation of immunodetection results. WB results showed occurrence of several proteins in protein pool of yeasts that were similar to staphylococcal proteins such as those with molecular weight of 57.5 and 66 kDa. Fluorescent microscopy showed interactions of FITC-antibodies with intracellular staphylococci which appeared as green spots. Hybridization of staphylococcal- specific probe with bacteria inside yeasts' vacuole confirmed immunodetection results. Detection of staphylococcal proteins and genes inside Candida albicans yeast indicates existence of intracellular bacteria inside the vacuole of yeast. These results suggest C. albicans as the potential reservoir of medically important bacteria.

Correlation of quinolone-resistance, qnr genes and integron carriage in multidrug-resistant community isolates of Klebsiella spp.

OBJECTIVES: Plasmid-mediated quinolone resistance (PMQR) determinants and integrons have a considerable contribution to bacterial drug resistance in Gram-negative pathogens. We studied the prevalence of PMQR genes and integron carriage in multidrug-resistant community isolates of Klebsiella spp. MATERIALS AND METHODS: Two hundred and fifty Klebsiella spp. isolates were collected from outpatient specimens between August 2015 and October 2017 in Yazd central Laboratory, Iran. Antibiotic susceptibility was determined against 17 antibiotics and minimum inhibitory concentration (MIC) of ciprofloxacin was measured by E-test. Polymerase chain reaction (PCR) was employed for detection of qnrA, qnrB, qnrS, aac(6')-Ib-cr, oqxAB and qepA genes. RESULTS: Disc diffusion results showed that 17 isolates (6.8%) were multidrug resistant (MDR), two of which were Klebsiella oxytoca and 15 were Klebsiella pneumoniae. MIC measurements revealed 11 ciprofloxacin-resistant isolates (including the two K. oxytoca), three intermediately-resistant and three ciprofloxacin-susceptible isolates. All ciprofloxacin-resistant and intermediately-resistant isolates carried at least one and up to four PMQR genes. The most prevalent PMQR gene was oqxAB (93.75%) followed by aac(6')-ib-cr (50.0%), qnrB (25.0%) and qnrS (18.75%) but qnrA and qepA were not detected. Class 1 integron was observed in 14 (82.3%) isolates including nine ciprofloxacin-resistant, two intermediately-resistant, and three susceptible isolates. Class 2 and 3 integrons were not observed. CONCLUSION: Presence of MDR, multiple PMQR determinants as well as class 1 integron in community isolates of Klebsiella spp. can be an important source of transmission of these opportunistic pathogens.

Candida albicans Release Intracellular Bacteria When Treated With Amphotericin B.

BACKGROUND: Coexistence of bacteria and yeast in a myriad of microbial communities is indicative of their intimate relationship, which could be the intracellular existence of bacteria inside yeast. In this study, the intracellular existence of bacteria inside yeast and bacterial release from amphotericin B-treated yeasts was examined using polymerase chain reaction (PCR) and microscopy. METHODS: Released bacteria, B1 from Y1 (a gastric yeast) and B2 from Y2 (an oral yeast) were identified as Staphylococcus hominis and Staphylococcus haemolyticus by biochemical tests as well as amplification of Staph-specific tuf and 16S rRNA genes. PCR products were sequenced and matched with Staphylococcus published sequences in GenBank. PCR was also used for amplification of Staph-tuf and Helicobacter pylori-16S rRNA genes from DNAs of 50 yeasts (20 oral, 20 gastric and 10 fecal). Microscopy was used for observing bacterium-like bodies (BLBs) inside the yeasts vacuole. RESULTS: Thirty-two yeasts (64%) carried Staph-tuf gene, 20 yeasts (40%) carried H. pylori 16S rRNA gene, 14 yeasts (28%) carried both genes, 12 yeasts (24%) carried neither, 6 yeasts (12%) carried only H. pylori 16S rRNA gene, and 18 yeasts (36%) carried only tuf gene. Amplified products of tuf (370 bp) and 16S rRNA (756 bp) genes from B1 and Y1, and B2 and Y2 showed high similarity to S. hominis and S. hemolyticus, respectively. Microscopic observations showed BLBs inside the yeasts vacuoles, which could be related to the released bacteria. These BLBs were alive and could be observed in successive generations of yeasts. CONCLUSION: Amplification of Staphylococcus- and H. pylori- specific genes from yeasts showed that the intracellular BLBs could belong to Staphylococcus species and H. pylori. Release of culturable staphylococci from 2/50 (4%) yeasts showed that not all yeasts release bacteria, and bacterial release takes place under unknown conditions. However, it could be triggered by amphotericin B or hydrolytic enzymes. Coexistence of staphylococci and H. pylori genes could represent a mixed endosymbiotic bacterial population in Fungi such as yeast. By selecting certain bacterial associates, the diversity of microbial communities could be determined. These selected bacteria could have an intracellular origin, being released under certain conditions.

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli.

BACKGROUND AND OBJECTIVES: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. MATERIALS AND METHODS: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. RESULTS: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient's serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. CONCLUSION: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis.

Extended-spectrum ß-lactamase and carbapenemase production among burn and non-burn clinical isolates of Klebsiella pneumoniae.

BACKGROUND AND OBJECTIVES: Klebsiella pneumoniae is an opportunistic pathogen responsible for up to 10% of nosocomial infections. The emergence and spread of multidrug resistant K. pneumoniae, mostly due to the production of extended-spectrum ß-lactamases (ESBL) and carbapenemases, is often responsible for antibiotic treatment failure of these infections. We compared the antibiotic resistance profiles, ESBL and carbapenemase production as well as presence of KPC-type genes in burn and non-burn clinical isolates of K. pneumoniae. MATERIALS AND METHODS: Fifty five clinical isolates were collected from Shahid Motahari (25 burn isolates) and Shariati (30 non-burn isolates) hospitals between August 2011 to January 2012. Antibiotic susceptibility was determined to 12 antibiotics using disc diffusion. The phenotypic confirmatory test (PCT) was used to screen for ESBL production. Carbapenemase activity was measured by the modified Hodge test (MHT) and KPC-type carbapenemases were further sought by PCR using specific primers. RESULTS: Both groups were highly resistant to cefotaxime and ceftazidime (>92%). Burn isolates were significantly more resistant to cefepime, amoxiclav, imipenem, meropenem, gentamicin and ciprofloxacin compared to the non-burn strains (p<0.05). No significant differences were observed in ESBL production between the two groups. Carbapenem resistance was only observed among the burn isolates (n=5, 9.1%). Five carbapenem-resistant isolates produced carbapenemases. However, none of the isolates harbored the KPC-type genes. CONCLUSION: Higher rates of drug resistance were observed in burn isolates of K. pneumoniae compared to the non-burn strains. Carbapenemase phenotype was only observed among the burn isolates but KPC-type gene was not detected.

Prevalence of qnr and aac(6')-Ib-cr Genes in Clinical Isolates of Klebsiella Pneumoniae from Imam Hussein Hospital in Tehran.

BACKGROUND: Plasmid mediated quinolone resistance (PMQR) has been shown to play an important role in resistance not only to quinolones, but also ß-lactams and aminoglycosides. In fact, qnr genes are frequently carried along with ß-lactamase determinants on the same plasmids. We studied the prevalence of qnrA, qnrB, qnrS and aac(6')-Ib-cr genes among quinolone and cephalosporin resistant clinical isolates of Klebsiella pneumoniae (K. pneumoniae), as well as the association between PMQR genes with resistance to quinolones, cephalosporins and aminoglycosides. METHODS: The study was conducted on 79 K. pneumoniae clinical isolates collected from Imam Hussein hospital in Tehran between July 2010 and January 2011, based on their resistance to quinilones and cephalosporins. Antibacterial susceptibility was determined to 15 antibiotics by disc diffusion. Presence of qnrA, qnrB, qnrS and aac(6')-Ib-cr genes were investigated using specific primers and PCR. RESULTS: Of the 79 K. pneumoniae isolates, 47 (59.5%) carried the PMQR determinants. Among these, 42 (89.4%) carried aac(6')-Ib-cr of which, 21 (50%) also harbored qnrB. Three isolates carried qnrB alone, two (4.2%) harbored qnrS and none had qnrA. Resistance to aminoglycosides and cephalosporins was significantly higher in the isolates carrying both qnrB and aac(6')-Ib-cr genes compared to aac(6')-Ib-cr alone. CONCLUSION: This study showed a high prevalence of aac(6')-Ib-cr and qnrB genes among the Iranian K. pneumoniae clinical isolates as well as co-carriage of the two genes. There was a significant association between qnrB gene carriage and resistance to quinolones, cephalosporins, and aminoglycosides.

Multidrug resistance and integron carriage in clinical isolates of Pseudomonas aeruginosa in Tehran, Iran.

BACKGROUND/AIM: Pseudomonas aeruginosa is the cause of 10% of hospital-acquired infections. The organisms are often multidrug- resistant, mediated mostly by antibiotic-resistant integrons. The aim of this research was to study integron carriage and its association with multidrug resistance in burn and nonburn clinical isolates of P. aeruginosa. MATERIALS AND METHODS: A total of 112 P. aeruginosa clinical isolates were collected from the Motahari and Shohadaye Tajrish hospitals in Tehran between July and December 2011. Antibiotic susceptibility to 13 antibiotics was determined by disk diffusion. Detection of integron classes 1 and 2 and amplifications of internal variable regions (IVRs) of class 1 integrons were carried out by PCR and specific primers. RESULTS: Among the 112 isolates, 77 were from burn patients and 35 were nonburn isolates. Multidrug resistance and class 1 integron carriage were both significantly higher in the burn isolates compared to the nonburn strains (97.4% vs. 22.8% and 82.3% vs. 17.7%, respectively). Class 2 integron (2.7%) was only present in the burn isolates. Amplification of IVRs of class 1 integrons revealed 3 different fragment arrays. CONCLUSION: The significant association between multidrug resistance and integron carriage among P. aeruginosa burn isolates suggests a dissemination of resistance determinants by horizontal gene transfer.

Correlation of Oxacillinase Gene Carriage With the Genetic Fingerprints of Imipenem-Resistant Clinical Isolates of Acinetobacter baumannii.

BACKGROUND: Emergence of multidrug-resistant Acinetobacter baumannii has resulted in the treatment failure of related infections and an increase in patient mortality. The presence of class D ß-lactamases (oxacillinases) in this organism is an important mechanism underlying resistance to beta-lactam antibiotics. OBJECTIVES: The aim of this work was to investigate the correlation between oxacillinase gene carriage and genetic fingerprints in imipenem-resistant burn and non-burn isolates of A. baumannii. MATERIALS AND METHODS: Fifty-eight A. baumannii isolates were collected from October 2011 to April 2012, which included 28 burn isolates from Shahid Motahari Hospital and 30 non-burn isolates from Imam Hossein Hospital. The minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) of imipenem were measured by the broth microdilution method. The presence of oxacillinase genes (OXA-23-, OXA-24-, OXA-51-, and OXA-58-like genes) was shown using type-specific primers and PCR. Genetic profiles were generated by RAPD-PCR fingerprinting. RESULTS: OXA-23 was observed in 81% of the isolates and its distribution was similar within the two groups. The presence of OXA-51 was shown in 58.6% of the isolates, of which most were burn isolates (67.6%). OXA-24 was present in 20.7% of the isolates, all belonging to the burn group; OXA-58 was not observed in any of the isolates. RAPD-PCR fingerprints revealed two clusters at a similarity level of 70% (A, B). At a similarity level of 85%, nine different groups were observed for burn and non-bun isolates. CONCLUSIONS: Our results showed that bla OXA-23 was the most prevalent gene, followed by bla OXA-51 , among the burn and non-burn clinical isolates of A. baumannii. Bla OXA-24 -like genes were detected at a lower level and were only found among the burn isolates, which also showed higher heterogeneity compared to the non-burn group.

Biofilm Formation and ß-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum ß-lactamases (ESBL), metallo ß-lactamases (MBL), and AmpC ß-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster. OBJECTIVES: The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC ß-lactamase production with biofilm formation among the isolates. MATERIALS AND METHODS: Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR. RESULTS: Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm. CONCLUSIONS: Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC ß-lactamase production. More importantly, multiple-ß-lactamase phenotype was associated with formation of strong biofilms.

Correlation of RAPD-PCR Profiles with ESBL Production in Clinical Isolates of Klebsiella pneumoniae in Tehran.

BACKGROUND: Multidrug resistant K. pneumoniae, particularly the extended-spectrum ß-lactamase (ESBL) producing strains, are often responsible for the failure of antibiotic treatment in nosocomial infections. Employing molecular methods to distinguish between ESBL and non-ESBL producing isolates can help quick identification of these multidrug resistant pathogens and thereby initiating appropriate antibiotic therapy. The aim of this study was to employ RAPD-PCR to distinguish the genetic fingerprints of ESBL producing clinical isolates of K. pneumoniae from ESBL negative strains. MATERIALS AND METHODS: Antibacterial susceptibility of 104 K. pneumoniae clinical isolates was determined to 13 antibacterial agents by disc diffusion. ESBL production was measured by the double disc synergy test followed by phenotypic confirmatory tests. Genetic fingerprinting was carried out by RAPD-PCR. RESULTS: All isolates were susceptible to imipenem. Antibiotic resistance rates were: piperacillin (100%), ceftazidime (62.5%), cefotaxime (57.6%), aztreonam (52.8%), cefepime (51.9%), kanamycin (50.9%), gentamicin (41.3%), ciprofloxacin (37.5%), nitrofurantoin (30.6%), nalidixic acid (22.1%), piperacillin/tazobactam (21.1%) and amikacin (9.6%). ESBL production was observed in 14 isolates (13.4%). Genetic fingerprinting performed on 43 isolates (14 ESBL positive and 29 ESBL negative) by RAPD-PCR, showed that 46.5% of the isolates belonged to a single profile (genotype 1), of which, the majority (62.1%) were non-ESBL producers. CONCLUSION: RAPD-PCR results showed heterogeneity among the isolates. There was no association between ESBL production with any specific genetic fingerprint.

Prevalence of Extended-Spectrum and Metallo ß-Lactamase Production in AmpC ß-Lactamase Producing Pseudomonas aeruginosa Isolates From Burns.

BACKGROUND: Pseudomonas aeruginosa is one of the most common causes of nosocomial infections. Resistance of P. aeruginosa to ß-lactam antibiotics may be the result of acquired resistance through mutation and over production of various antibiotic inactivating enzymes. This research aimed to determine the prevalence of extended-spectrum ß-lactamases (ESBL) and metallo ß-lactamase (MBL) production as well as the presence of their related genes among AmpC ß-lactamase producing P. aeruginosa isolated from burns. OBJECTIVES: The current study aimed to determine the prevalence of class A ESBL and MBL production in relation to the presence of their related genes among AmpC ß-lactamase producing P. aeruginosa isolated from burns. MATERIALS AND METHODS: The antimicrobial susceptibility of 51 P. aeruginosa isolates from patients with burns was examined against 13 antibiotics by the disc diffusion method. Minimum inhibitory concentrations (MIC) for imipenem and ceftazidime were measured by the microdilution method. AmpC production was detected by AmpC disc and the modified three-dimensional extract tests. ESBL phenotype was confirmed by the double disc synergy test (DDST). Presence of ß-lactamase genes was detected by specific primers and polymerase chain reaction (PCR). RESULTS: All isolates were multidrug resistant. AmpC, ESBL and MBL production were observed in 35 (68.6%), 20 (39.2%) and 19 (37.3%) isolates, respectively. Overall, 43 isolates (84.3%) carried ß-lactamase genes, out of which 31 (60.8%) harbored bla AmpC , 20 (39.2%) had bla TEM and 11 (21.6%) carried bla PER -1 genes. Among the AmpC producers, two isolates (6.5%) carried bla AmpC + bla ESBL , 13 (41.9%) had bla AmpC + bla MBL and six (19.4%) produced the three enzymes. CONCLUSIONS: A high prevalence of multiple ß-lactamase production was observed among the AmpC producers (60%), of which the majority co-produced AmpC and MBL. The current study results showed correlation between ß-lactamase production and the presence of antibiotic resistance genes in the isolates.

Prevalence of Quinolone Resistance Among Extended-Spectrum ß -Lactamase Producing Uropathogenic Klebsiella pneumoniae.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL) production is the major resistance mechanism to ß-lactam antibiotics in Enterobacteriaceae. In addition, emergence of plasmid-mediated quinolone resistance (PMQR) in ESBL-producing isolates has become a global threat for treatment of these infections. OBJECTIVES: We investigated the association between ESBL production and quinolone resistance in urinary isolates of K. pneumoniae. PATIENTS AND METHODS: A total of 196 urinary isolates of K. pneumoniae were collected from Imam Hussein Hospital in Tehran during a four year period (2008-2012). Antibiotic susceptibility was determined by disc diffusion and ESBL production was screened using the phenotypic confirmatory test (PCT). RESULTS: All isolates were susceptible to imipenem. Resistance to piperacillin and cefotaxime were 66.3% and 50.5%, respectively. Resistance to ceftazidime, amoxiclave, aztreonam, ceftriaxone, cefepime, nitrofurantoin, gentamicin, ciprofloxacin, nalidixic acid, ofloxacin, norfloxacin, levofloxacin, amikacin and pipracilin/tazobactam were less than 50%. ESBL production was detected in 92 isolates (46.9%) of which, 61.9% were resistant to nalidixic acid and 65.2% to ciprofloxacin. Multidrug-resistance was observed in 96.7% of ESBL producers. CONCLUSIONS: Our results showed coexistence of ESBL and quinolone resistance in the majority of the uropathogenic K. pneumoniae test isolates suggesting that care should be taken for the choice of antibiotic therapy.

Quinolone Susceptibility and Detection of qnr and aac(6')-Ib-cr Genes in Community Isolates of Klebsiella pneumoniae.

BACKGROUND: Plasmid-mediated quinolone resistance genes (PMQR) have been shown to play not only an important role in quinolone resistance, but also resistance to other antibiotics, particularly ß-lactams and aminoglycosides. These genes are mainly associated with clinical isolates of Enterobacteriaceae. However, detection of PMQR genes in the community isolates can increase the dissemination rate of resistance determinants among bacteria. OBJECTIVES: This study aimed to investigate quinolone resistance and distribution of qnr and aac (6')-Ib-cr genes among the community isolates of Klebsiella pneumoniae. MATERIALS AND METHODS: Fifty-two K. pneumoniae isolates were collected from the Central Laboratory in Karaj between July 2010 and January 2011. Antibacterial susceptibility was determined by the disc diffusion method. Quinolone and/or cephalosporin-resistant isolates were screened for the presence of qnrA, qnrB, qnrS and aac (6')-Ib-cr genes by polymerase chain reaction (PCR). RESULTS: Of the 52 K. pneumoniae isolates, 23 were resistant to cephalosporins and/or quinolones. Overall, 7 out of the 23 resistant isolates harbored qnr and/or aac (6')-Ib-cr genes (30.4%). Among these, 5 isolates were resistant to both classes of antibiotics of which; 3 carried the aac (6')-Ib-cr gene, one had the qnrS, and one harbored both aac (6')-Ib-cr and qnrB genes. None of the isolates contained qnrA. Two isolates were sensitive to quinolones and resistant to cephalosporins of which; one had qnrS and the other carried the aac (6')-Ib-cr gene. CONCLUSIONS: Our study showed that 30.4% of the quinolone and/or cephalosporin resistant community isolates of K. pneumoniae carried PMQR genes. These results confirm that community isolates can be an important source for spreading antibiotic resistance determinants among Gram negative pathogens. This is the first report from Iran on detection of PMQR in the community isolates of K. pneumoniae.

Correlation of Multi-drug Resistance, Integron and blaESBL Gene Carriage With Genetic Fingerprints of Extended-Spectrum ß-Lactamase Producing Klebsiella pneumoniae.

BACKGROUND: Some genetic and phenotypic variables are associated among distinct microbial populations. OBJECTIVES: The associations between multi-drug resistance (MDR) phenotypes, prevalence of antibiotic resistance integrons (ARIs), bla SHV, bla TEM and bla CTX-M gene carriage and genetic fingerprints of random amplified polymorphic DNA (RAPD), confirmed by pulsed field gel electrophoresis (PFGE), were investigated among extended-spectrum ß-lactamases (ESBL)-producing nosocomial isolates of Klebsiella pneumoniae. MATERIALS AND METHODS: Susceptibility of 35 ESBL-producing K. pneumoniae nosocomial isolates to 22 antimicrobial agents was determined. Integron carriage was detected using specific primers for intI1, intI2 and intI3 genes by PCR. RESULTS: All isolates were resistant to piperacillin and susceptible to imipenem. MDR phenotype was observed in 91.4% of the isolates. Class 1 integrons were detected in 21 (60%) and class 2 integrons in 3 (8.57%) of the isolates. Two of the isolates carried both classes and none harbored class 3 integrons. Significant correlations were observed between resistance to aminoglycosides, fluoroquinolones and sulfonamides, and between genotype groups with carriage of ARIs, MDR phenotype and bla SHV gene carriage. ARI carriage was also significantly associated with MDR phenotype. CONCLUSIONS: Our findings suggest the possible co-carriage of some bla SHV genes and ARIs on the same plasmids harboring the MDR genes. Possible role of integrons in dissemination of ESBL-encoding bla SHV genes among ESBL-producing K. pneumoniae nosocomial isolates may be inferred.

Prevalence of blaIMP, and blaVIM gene carriage in metallo-ß-lactamase-producing burn isolates of Pseudomonas aeruginosa in Tehran.

BACKGROUND/AIM: To study the prevalence of blaVIM and blaIMP genes in metallo-ß-lactamase (MBL)-producing burn isolates of Pseudomonas aeruginosa in relation with AmpC and extended-spectrum ß-lactamase (ESBL) production. MATERIALS AND METHODS: Thirty-two carbapenem-resistant MBL-producing P aeruginosa burn isolates from Shahid Motahari Burn Hospital in Tehran were employed. Antibiotic susceptibility was determined to 13 antibiotics including imipenem and meropenem by disk diffusion. AmpC and ESBL production was detected by the AmpC disk test and combined disk diffusion assay, respectively, blaIMP and blaVIM gene carriage was shown by polymerase chain reaction and type-specific primers. RESULTS: AmpC production was observed in 81% and ESBL production was detected in 12.5% of the isolates. blalMP carriage was observed in 56.25% and blaVIM gene in 46.8% of the isolates. Surprisingly, 43.5% of the isolates carried both blalMP and blaviM genes. CONCLUSION: We think that this is the first report on the cocarriage of blalMP and blavIM in P aeruginosa. There was also a strong association between MBL gene carriage and AmpC ß-lactamase production.

Genetic profiling of Klebsiella pneumoniae: comparison of pulsed field gel electrophoresis and random amplified polymorphic DNA

In this study, the discriminatory power of pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods for subtyping of 54 clinical isolates of Klebsiella pneumoniae were compared. All isolates were typeable by RAPD, while 3.6% of them were not typeable by PFGE. The repeatability of both typing methods were 100% with satisfying reproducibility (≥ 95%). Although the discriminatory power of PFGE was greater than RAPD, both methods showed sufficient discriminatory power (DI > 0.95) which reflects the heterogeneity among the K. pneumoniae isolates. An optimized RAPD protocol is less technically demanding and time consuming that makes it a reliable typing method and competitive with PFGE.