Molecular characterisation of Klebsiella oxytoca strains isolated from patients with antibiotic-associated diarrhoea

Background and study aim: Colitis is a common complication after treatment with antibiotics such as beta-lactarns, quinolones, and aminoglycosides. Recently, Klebsiella oxytoca has been implicated in this type of diarrhoea. The prevalence and characterisations of K. oxytoca isolated from patients with antibiotic-associated diarrhoea were investigated. The K. oxytoca isolates were also tested for cytotoxin production

Patients and methods: This study was conducted from May 2011 to Dec 2013. Faecal samples were collected from hospitalised patients receiving antibiotic treatment. Initial cultivation was performed on specific media. The clinical isolates were confirmed by poIymerasc chain reaction [PCR] using the specific K. oxytoca po1ygalacturonase [pehX] gene. The double-disc diffusion test was used to detect extended-spectrum beta-lactamase [ESBL]-producing strains. Tracking of ESBL-encoding genes was performed via PCR. The organism was cultured on Hep-2 cell lines for cytotoxin production

Results: Out of 331 samples collected from patients, 40 were confirmed molecularly to be clinical isolates of K. oxytoca. Fourteen [35%] ESBL-producing strains were isolated using the double-disc diffusion method. Among the molecularly confirmed K. oxytoca isolated seven [17.5%] tested positive for the blaSHV gene, 12 [30%] for blaTEM, 10 [25%] for blaCTX-M, three [75%] for blaOXA, nine [22.5%] for blaCTX M-15, and seven [17.5%] for blaTEM-1. Five [12%] isolates showed cytotoxin activity below 30%, 12 [30%] strains showed moderate cytotoxin activity between 30% and 60%, and 23 [58%] strains showed cytotoxin activity >/=60%

Conclusions: the cytotoxin-producing K.oxytoca is found to be one of the causes of antibiotic-induced colitis. Discontinuing treatment and allowing normal intestinal flora to be established or prescribing appropriate medication after antibiogram can help patients with antibiotic-induced haemorrhagic colitis in a timely manner

Genetic profile variation in vaccine strains and clinical isolates of bordetella pertussis recovered from Iranian patients

Re-emergence of pertussis has been reported in Iran despite a high rate of vaccination coverage. Low efficacy of the vaccine might be due to the genetic divergence between Clinical versus vaccine strains. In the current study, the genetic profiles of Clinical isolates and vaccine strains of Bordetella pertussis [B. pertussis] were assessed by using Pulsed Field Gel Electrophoresis [PFGE]. Following phenotypic and molecular identification of isolates, XbaIdigested genomic DNA of 5 Clinical isolates, 2 vaccine strains and a Tohama I strain were analyzed by PFGE along with B. parapertussis as a control. Seven distinct PFGE profiles were found among all examined isolates/strains. In 5 Clinical isolates, 4 profiles were identified whereas the vaccine strains displayed 2 distinct profiles. The reference strain, Tohama I had a distinct profile. Vaccine and Clinical profiles had low similarity, with relatedness of approximately 40%. The genetic profiles of B. pertussis were different between circulating isolates and vaccine strains used in the national vaccination programs. Since new genetic profiles of B. pertussis can be disseminated periodically, the profiles of isolates circulating in the population should be monitored over the course of the re-emergence

[Identification of bla-CTX-M beta-lactamase in Klebsiella pnumoniae clinical isolates by polymerase chain reaction]

The incidence of extended-spectrum beta- lactamase- producing bacteria has been increased worldwide. The most common cause of resistance to extended-Spectrum cephalosporins in Klebsiella pneumoniae is the production of extended-spectrum beta lactamases [ESBLs]. In the past decade, CTX-M enzymes have become the most prevalent ESBLs in Europe, Canada and Asia. The aim of this study was to find the prevalence of ESBL-producing K .pneumoniae and molecular detection of CTX-M group in these bacteria. In the descriptive study, 100 K. pneumoniae isolates were detected bystandard biochemical tests between April 2012 and September 2012, in Besat and Imam Reza hospitals of Tehran, Iran. Susceptibility to antimicrobial agents was tested for 10 antibiotics by the disk agar diffusion [DAD] method. Also, ESBL production was screened by combined disk diffusionas recommended by the Clinical and Laboratory Standards Institute [CLSI]. Then, Screened isolates were assayed by PCR for detection of CTX-M-1 group genes. Of 100 K. pneumonia isolates, 26 isolates produced ESBLs, and also 42 isolates were CTXM- 1 producer using PCR method. According to the differences between the results of phenotypic and genotypic tests, it seems that molecular detection of drug-resistance genes is necessary. Further investigations are needed to determine the epidemiology of ESBL producing K. pneumoniae in Iran

Molecular characterization of class 1 integrons and gene cassettes in multidrug resistant [MDR] Klebsiella spp. isolated from hospitalized and outpatients in Iran, 2009

Klebsiella species are of the most common bacteria involved in nosocomial and urinary tract infections. Genetic elements such as class 1 integrons have an important role in the resistance development. In this study, the share of class 1 integrons, the genetic characterization of the integron cassettes and PFGE profiles of the clinical Klebsiella isolates are evaluated in Besat University hospital of Sanandaj, Iran. Isolates from 17890 clinical specimens were identified byAPI20E. Antibiotic susceptibility testing MIC were done for MDR isolates. For investigating class 1 integrons and gene cassettes, PCR by inti1 integrase and 5-CS/3-CS were performed. Integrated gene cassettes were analyzed by PCR-RFLP and sequencing. Pulsed-Field Gel Electrophoresis was carried out for studying of clonality outbreak of isolates. Thirty five Klebsiella spp. were isolated and included 29 K. pneumoniae and six K. oxytoca. All the isolates were susceptible to carbapenems while other antibiotics showed high resistant profile. In all Klebsiella spp. PCR for intl1 integrase and 5-CS/3-CS were positive [100%]. Sequencing for prevalent bands of internal variable regions between 5-CS/3-CS showed arr-5, orfD-aacA4 and aad5- dfrA 17. PFGE Analysis showed 18 clusters in K. pneumoniae with clonality relatedness in some cases but no relatedness among K. oxytoca isolates. High prevalence of class 1 integron carrying gene cassettes confirms that integron-mediated antimicrobial gene cassettes are important in Klebsiella spp. resistance profile. Clone diffusions of MDR Klebsiella spp. which harbor class 1 integrons have threaten the potential in the resistance development in our clinical settings

Metallo- beta-lactamases among multidrug resistant [MDR] gram negative bacteria isolated from clinical specimens during 2009 in Sanandaj, Kurdistan province

Today, there are numerous reports about emerging multi drug resistant gram negative bacteria all around the world, especially in ICUs. Rarely, Metallo-beta-lactamase [MBL] enzymes are responsible for these cases. Study of MBLs for diagnosing and preventing distribution of the origin of infection are critical issues. In addition, we would like to compare the efficacy of Iranian and foreign- made antibiotic disks. During 2009 all entered clinical specimens to the laboratory tested for detecting gram negative bacteria. Isolated bacteria were tested by Kirby-Bauer method to antibiotic susceptibility test by Iranian and foreign [MAST] disks. For gram negative carbapenem resistant isolates, PCR technique used to detect VIM, GIM, and SIM variants of MBLs. During one year, 17890 clinical specimens referred Besat laboratory. The most specimen was Urine [8172] followed by blood culture [5190] that in which 1110 gram negative and positives isolated. Out of which, 778 [70%] of isolates were gram negatives. MDR gram negatives were 157 [20.2%]. Imipenem and meropenem were the most efficient antibiotics [all susceptible] and ceftriaxone was the least [19% susceptible]. E. coli was the most prevalent isolate. 79 Gram negative isolates [10.1%] were resistant to Iranian-made discs but all susceptible for foreign ones. All 79 isolates were tested by PCR for MBL genes, that, all were negative. Besides, Iranian imipenem and cefepime disks have had distinguishable difference in susceptibility of isolates. Fortunately, none of gram negative isolates were MBL producer, which revealed no colonization of MBL producing bacteria. Iranian-made disks appear efficient except for imipenem and cefepime.

[Molecular detection and identification of virulence factors of Pseudomonas aeruginosa isolated from wound and burn infections]

In recent decades, Pseudomonas aeruginosa has emerged as one of the most important nosocomial pathogens. Due to the clinical importance this bacterium, various methods have been developed to rapidly and accurately identify it. The aim of this research was to detect P. aeruginosa isolated from wound and burn infections on the basis of the amplification of the oprl, oprL and toxA genes, and to determine the prevalence of nanl and exoS genes among them. A total of 150 P. aeruginosa isolates was collected from patients with burn and wound infections of Imam-Khomaini, Tohid and Motahari hospitals in Tehran. The isolates were identified as P. aeruginosa using specific biochemical tests. Chromosomal DNA of the isolates was extracted with phenol chloroform method and used for PCR of oprl, oprL, toxA, exoS and nanl genes by specific primers. Among 150 P. aeruginosa isolates all carried the oprl and oprL genes; 98 [65.3%] 142 [94.7%] and 19 [12.66%] of the isolates were positive for exoS, toxA and nanl genes respectively. The presence of nanl gene in wound isolates [30%] was significantly higher [p<0.05] than in burn isolates [4%].Our results indicated that simultaneous use of oprl, oprL and toxA genes provide sufficient sensitivity to detect P. aeruginosa in clinical samples. The high prevalence of exoS in isolates suggests invasive phenotype of wound and burn isolates. The high prevalence of nanl in wound isolates suggests a possible role of this gene in those infections

cholera outbreak associated with drinking contaminated well water

Cholera has been a significant public health challenge in many communities. An outbreak of acute diarrheal illness occurred among participants in a wedding ceremony in a village in Qazvin, Iran, in 2008. We conducted an epidemiological, environmental and microbiological investigation to determine the causative agent, source and extent of this outbreak. Clinical and environmental samples were collected and analyzed for the presence of diarrhea-causing bacterial organisms, which included Vibrio cholera. The relationship between the strains was determined using enterobacterial repetitive intergenic consensus poly-merase chain reaction [ERIC-PCR]. The attack rate was 21.8%. Clinical and environmental samples were positive for V. cholerae serotype Inaba. All tested isolates had a similar ERIC-PCR pattern, which indicated that a single clone of V. cholerae was responsible for this outbreak. Our findings demonstrated that well water was the source of this outbreak

PER, CTX-M, TEM and SHV beta-lactamases in clinical isolates of klebsiella pneumoniae isolated from Tehran, Iran

Different types of extended spectrum beta-lactamases [ESBLs] are encountered in the clinical settings worldwide. There are a few studies regarding the prevalence of ESBL genes among Klebsiella pneumoniae isolates at Tehran especially those of bla[PER] and bla[CTX]. The aim of this study was to determine the prevalence of bla[SHV], bla[TEM], bla[PER] andbla[CTX] genes among clinical K. pneumoniae of different hospitals in Tehran. Two hundred isolates of K. pneumoniae were received from different clinical specimens. The susceptibility of the isolates to 10 different antibiotics was examined by disk diffusion test. The MICs for ceftazidime were also determined using micro-broth dilution assay. Isolates showing MIC >/= 4 micro g/ml for ceftazidime were screened for ESBL production by phenotypic confirmatory test [PCT] and subjected to PCR for studied genes. Variation among four amplified genes was evaluated using PCR-RFLP. By disk diffusion test, resistance to ceftazidime and cefotaxime were 34.7% and 33.5% respectively. However, all strains were susceptible to imipenem. Eighty isolates showed MICs >/= 4 micro g/ml for ceftazidime of which 77 [96%] were positive for ESBL in PCT. The prevalence of bla[SHV], bla[CTX-M], bla[TEM] and bla[PER] among these isolates were 26%, 24.5%, 18% and 7.5%, respectively. No variation was detected in the genes by PCR-RFLP. As far as we know this is the first report of the bla[PER-1] in K. pneumoniae in Iran. The bla[CTX-M] was the second most common gene detected among the ESBL positive isolates of K. pneumoniae. For rapid identification of ESBL producing isolates it was recommended that clinical laboratories adopt simple test based on CLSI recommendation for confirming ESBL production in enterobacterial species

survey of genes encoding beta-lactamases, in escherichia coli resistant to beta-lactam and non-beta-lactam antibiotics

Resistance to the new generation of cephalosporins which is mediated by Extended-Spectrum beta-lactamases [ESBLs] has been found among Escherichia coll isolates throughout the world. These resistance genes and their producers, the micro-organisms carrying beta-lactamases, are responsible for serious clinical and therapeutic problems among inpatients and it is necessary to pay more attention to detection of ESBLs producing organisms. Collectively 260 isolates of E. coli were obtained from 6 hospitals in Tehran [Iran] during April-2006 to April-2007. The antibiotic susceptibility patterns of isolates were determined by disk diffusion method phenotypic confirmatory test [PCT] was carried out for screening of ESBLs. Microbroth dilution assay was used to determine the minimum inhibitory concentration [MIC] of ceftazidime. Isolates showing MIC >/= 2 microg/ml were subjected to polymerase chain reaction [PCR] targeting bla[TEM], bla[SHV], bla[CTX] and bla[PER] genes. The PCT showed that 48.08% of isolates are ESBL producers [125 of 260]. The majority of cefotaxime resistant [90.8%] and ceftazidime resistant [92, 5%] isolates were ESBL producers. The obtained results by PCR revealed that 5.77% [n=15 of 260] and 24.23 [n=63] of isolates can produce SHV and TEM type enzymes respectively. Bla[CTX] was detected in 20.38% of isolates [n=53] and none of them could produce bla[PER] type beta-lactamases. The results of our study showed that the ESBL genes have high prevalence among clinical isolates of E. coli. Such high dissemination of ESBLs is a serious problem for public health and therefore, it's necessary to seek a program for monitoring ESBLs in hospitals

Detection of TEM, SHV and PER type extended-spectrum B-Lactamase genes among clinical strains of pseudomonas aemginosa isolated from burnt patients at Shafa-Hospital, Kerman, Iran

This study was carried out to evaluate the existence of the TEM, SHY and PER ESBL genes in ESBL producing strains of Pseudomonas aeruginosa isolated from burnt patients at Shafa-hospital, Kennan, Iran. A total of 120 strains of P. aeruginosa were isolated from 245 patients in burn unit of Shafa-hospital during January 2006 to December 2007. MIC of antibiotics was measured using agar dilution test ESBL producing strains were detected by double-disc synergy method containing amoxicillin and amoxicillin+clavulanic acid and phenotypic confirmatory test. All the clinical isolates resistant to imipenem [IMP] were screened for the production of MBL by E-test with IMP/IMP+EDTA strips. PCR and multiplex-PCR performed for the detection of different types of ESBL producing genes in ESBL positive isolates. Of 120 the isolates, 3-5% showed MIC greater than 16 mg/ml to IMP and meropenem, 66% showed MIC greater than 32 mg/ml to ceftazidime, 42% to azteronam and 60% of the isolates showed MIC greater than 64 mg/ml to cefotaxime, 41 [34%] contlnned as ESBL producers. Not any isolate could produce MBL [pœ0.05]. The PCR assay of all ESBL producing isolates revealed that 6.6%, 4.1% and 2.5% of them were positive for SHY, PER and TEM genes, respectively. Many ESBL producing strains of P. aeruginosa isolated from patients in burn unit of Shafa-hospital. However, none could produce MBL enzyme. The genes among ESBL producing strains were SHY, PER as well as TEM type of b-lacatamases

Bactericidal effects of essential oils from clove, lavender and geranium on multi-drug resistant isolates of Pseudomonas aeruginosa

The inhibitory effects of essential oils including clove, lavender and geranium extracted from Eugenia caryophyllata, Lavandula officinalis and Pelargonium graveolens on multi-drug resistant isolates of Pseudomonas aeruginosa were investigated. The main constituents of clove, lavander and geranium oil were eugenol [80-90%], 1,8-cineol [13%] and citronellol [45%] respectively. Clove had the most effective essential oil against P. aeruginosa. A combination consisting of clove, lavender and geranium oils at a ratio of 3:1:1 showed the most inhibitory effect [32-64 microg/ml] and strong synergy with gentamicin. The essential oils from clove, lavender and geranium exhibited bactericidal activity against multi-drug resistant strains of P. aeruginosa and may be alternatives compounds against these strains in the future

Isolation and purification of major outer membrane proteins from brucella abortus s-99

Isolation and purification of major outer membrane proteins [OMP] from the cell wall envelope of Brucella abortus S-99 were achieved by sonication, solubilization and membrane fractionation in the presence of non-ionic detergent [Tx-100] and lysozyme treatments, followed by ultracentrifugation. The crude OMP was treated with trypsin to free the preparation from any other protein contaminants. The OMP preparation was purified by column chromatography on Sephacryl S-200. Three major symmetrical peaks emerged from the column with kav values of 1.81, 2.42 and 2.56 in succession in addition to a few closely related minor peaks. Characterization of crude OMP on SDS-P AGE showed 13 protein bands. The three major peaks 1,2 and 3 were subjected to SDS-P AGE separately and the molecular weights of peaks 2 and 3 were calculated to be 26 and 38 kDa, respectively and the first peak was further resolved into two subfractions with molecular weights of 62 and 67 kDa. However, after treatment of OMP with trypsin the number of bands were reduced to one prominent band with a molecular weight of 38 kDa and a thinner band of 41 kDa