Airborne bacterial and PM characterization in intensive care units: correlations with physical control parameters.

In this study, the spatial variation of airborne bacteria in intensive care units (ICUs) was characterized. Fine particulate matter and several physical parameters were also monitored including temperature and relative humidity. The results showed that the total bacterial load ranged between 20.4 and 134.3 CFU/m3 across the ICUs. Bacterial cultures of the collected samples did not isolate any multi-drug-resistant Gram-negative bacilli indicating the absence of such aerosolized pathogens in the ICUs. Meanwhile, particulate matter levels in several ICUs were found to exceed the international guidelines set for 24-h PM exposure. Moreover, examining bacterial load contribution by size suggested that bacteria with sizes less than 0.65 µm contributed the least to the total bacterial loads, while those with sizes between 0.65 and 1.1 µm contributed the most. A multiple linear regression model was also built to predict the bacterial loads in the ICUs. The regression analysis explained 77% of the variability observed in the measured bacterial concentrations. The model showed that the level of activity in the ICU rooms as well as its occupancy level had strong positive correlations with bacterial loads, while distance away from the patient had a non-linear relationship with measured loads. No statistically significant correlation was found between bacterial load and particulate matter concentrations.

First detection of oxacillinase-mediated resistance to carbapenems in Klebsiella pneumoniae from Morocco.

The frequency of carbapenem resistance due to class-D beta-lactamases (i.e. oxacillinases) among the world's Enterobacteriaceae is increasing. Recently, in Morocco, two isolates of carbapenem-resistant Klebsiella pneumoniae were recovered from the same patient, one harbouring plasmid-encoded bla-(OXA-48) and the other the bla-(OXA-1) gene. This represents the first evidence of bla(OXA-48)-mediated carbapenem-resistance in Enterobacteriaceae in Morocco.

Molecular characterisation of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon.

The prevalence of bla CTX-M, bla TEM and bla SHV genes among extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Escherichia coli (n = 50) and Klebsiella spp. (n = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla CTX-M-15, bla TEM-1, bla OXA-1 and six bla SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.

Genomic Characterization of MDR Escherichia coli Harboring blaOXA-48 on the IncL/M-type Plasmid Isolated from Blood Stream Infection.

Escherichia coli is responsible for a wide variety of community and hospital acquired extraintestinal infections, and the emergence of ESBL resistant isolates is a major clinical concern. In this study, we characterized the genomic attributes of an OXA-48 and CTX-M-3 producing E. coli EC-IMP153. Whole-genome initial assembly produced 146 contigs with a combined 5,504,170 bp in size and a G+C content of 50.5%. wgSNPs-based phylogenetic comparison with 36 publically available genomes was also performed. Comprehensive genomic analysis showed that EC-IMP153 belonged to sequence type ST-405 and harbored several resistance determinants including the ß-lactam resistance genes blaOXA-48, blaCTX-M-3, blaTEM-1B, blaOXA-1, and blaCMY-70, aminoglycoside fyuA and aac(3)IId, tetracycline tet(A) and tet(R), and fluoroquinolone gyrA, parC, and mfd resistance determinants. Plasmids with the following incompatibility groups were detected in silico and confirmed using PBRT: IncI1-α, IncL, IncW, Col (BS512), and IncF. To our knowledge this is the first in-depth genomic analysis of an OXA-48 producing E. coli ST-405 isolated from a patient in Lebanon and linked to a blood stream infection. Continuous monitoring is necessary to better understand the continued diffusion of such pathogens, especially in view of the population movements triggered by unrest in the Middle East.

Immunoaffinity purification, stabilization and comparative characterization of listeriolysin O from Listeria monocytogenes serotypes 1/2a and 4b.

We developed a simple and highly effective procedure for stabilizing the haemolytic activity of listeriolysin O (LLO) from Listeria monocytogenes after immunoaffinity purification. The haemolytic activity of LLO was stabilized by eluting it directly into tubes containing an alkaline buffer (5 mM lysine, 140 mM KCl, 50% ethylene glycol, pH 11.5). The purified LLO retained 100% of its haemolytic activity after 6 weeks of storage at -20 degrees C. LLO purified from a strain of L. monocytogenes serotype 1/2a (ATCC 43249) and LLO purified from a strain of L. monocytogenes serotype 4b (F 2365) isolated from a Mexican-style cheese, showed no significant differences in pH and temperature stability. When incubated in buffers at pH values from 4 to 12 at 4 degrees C and 25 degrees C, LLO from serotypes 1/2a and 4b retained maximal haemolytic activity at pH 8 after 4 h of incubation. LLO from both serotypes lost their haemolytic activity after incubation at 50 degrees C for 25 min.

Bacterial etiology of otitis media with effusion in a group of Lebanese children.

Identification of the main bacteria causing otitis media with effusion (OME) in a given population is essential. It indicates the degree of involvement of a given bacterium in a particular disease of that population. Knowledge of the most prevalent bacteria would initiate the search for the mode of acquisition of such bacteria and may aid in establishing appropriate control and prevention programs, which may decrease the incidence of OME. The rapid response of most OME to a variety of broad-spectrum antimicrobials deprives the clinician from knowing the particular bacteriologic agent prevailing in a community. With the emergence of resistant strains and the change over time of the relative distribution of bacteriologic agents known to cause OME, the identification of the bacterial etiology of OME in Lebanese children was initiated.

Underlying mechanisms of carbapenem resistance in extended-spectrum ß-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: role of OXA-48 and NDM-1 carbapenemases.

A recent increase in carbapenem resistance among extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli isolates at a major tertiary care centre in Lebanon prompted the initiation of this study. Consecutive ESBL-producing isolates were tested for resistance to carbapenems, with initial screening by disk diffusion and Etest using ertapenem. The modified Hodge test was also performed. PCR of ß-lactamase-encoding genes, including bla(NDM-1), bla(KPC), bla(OXA-48), bla(CTX-M), bla(TEM), bla(SHV), bla(CMY-2) and bla(OXA-1), as well as outer membrane porin genes (ompC and ompF) was performed. Sequencing, efflux pump inhibitor tests and random amplified polymorphic DNA (RAPD) analysis were performed. In total, 14 (2.45%) of 572 K. pneumoniae and 24 (1.07%) of 2243 E. coli were ertapenem-non-susceptible [minimum inhibitory concentration (MIC) ≥0.25 µg/mL]. Resistance to other carbapenems was variable. PCR and sequencing analysis revealed that isolates harboured different ß-lactamase genes, including bla(OXA-1), bla(CTX-M-15), bla(TEM-1), bla(CMY-2), bla(OXA-48) and bla(NDM-1). In addition, K. pneumoniae lacked the outer membrane porin-encoding genes, whilst E. coli harboured them with detected mutations. CTX-M-15 was carried on a 90 kb plasmid, whilst OXA-48 was carried on a 70 kb plasmid. Efflux pump inhibition significantly decreased MICs in E. coli. RAPD analysis demonstrated genomic variability. In conclusion, carbapenem resistance in ESBL-producing K. pneumoniae and E. coli is due to the combined effect of ß-lactamases with porin impermeability and/or efflux pump activity observed in these organisms, and in a number of isolates is due to the production of the carbapenemase-encoding genes bla(OXA-48) and the newly emerging bla(NDM-1).

First detection and sequence analysis of the bla-CTX-M-15 gene in Lebanese isolates of extended-spectrum-beta-lactamase-producing Shigella sonnei.

The emergence in Shigella species of extended-spectrum beta-lactamases (ESBL) that impart resistance to third-generation cephalosporins is a growing concern world-wide. So far, however, ESBL-producing Shigella have only been reported seven times, albeit from seven different countries. In Lebanon, three ESBL-producing clinical isolates of S. sonnei were recovered from 30 cases of shigellosis diagnosed between July 2004 and October 2005. All three were found to be resistant to amoxycillin, cefotaxime, ceftazidime, aztreonam, trimethoprim/sulphamethoxazole, gentamicin, and kanamycin. Each harboured the bla-CTX-M gene, and the results of sequence analysis indicated this to be of the bla-CTX-M-15 type and encoded on a 70-kb plasmid, flanked by an insertion element (ISEcp1). The bla-TEM-1 gene was also detected on the chromosomes of two of the ESBL-producing isolates. Class-2 integrons containing dhfr1, aadA1 and sat1 genes were detected on the chromosomes of all three isolates but not on the plasmids. Fluoroquinolone-modifying factors [QnrA, QnrB, QnrS or AAC(6')-Ib-cr] were not detected. The results of RAPD analysis, combined with data on antimicrobial susceptibility, indicated that each isolate was unique. In conclusion, the emergence of ESBL-producing isolates of S. sonnei has been demonstrated for the first time in Lebanon. The resistance of these isolates to third-generation cephalosporins was mediated by the CTX-M-15 enzyme, which was plasmid-encoded.

Prevalence of the genes encoding extended-spectrum beta-lactamases, in Escherichia coli resistant to beta-lactam and non-beta-lactam antibiotics.

The prevalences of extended-spectrum beta-lactamases (ESBL) and their encoding bla genes, TEM, SHV and CTX_M, were investigated in isolates of Escherichia coli that were resistant to beta-lactam and/or non-beta-lactam antibiotics. Of the 250 E. coli isolates investigated, all of which came from patients in a major hospital in southern Lebanon, 61 (13.3%) were found to have ESBL, their production of beta-lactamase being confirmed by the ceftazidime and ceftazidime/clavulanic-acid disc methods. All 61 ESBL isolates were resistant to beta-lactams and sensitive to imipenem, piperacillin/tazobactam and cefoxitime. Only 40% were resistant to fluoroquinolones, 33% were resistant to aminoglycosides, and 18% were considered to have multi-drug resistance. The results of the PCR-based amplification of the bla gene in DNA samples from the 61 ESBL isolates indicated that 11 (18%) of the isolates carried both the TEM and SHV genes, 37 (61%) carried the TEM gene but not the SHV, and 13 (21%) had the SHV gene but not the TEM. None of the isolates carried the CTX_M gene. Of the 37 TEM-positive/SHV-negative isolates, 43% were resistant to fluoroquinolones and 37% to aminoglycosides. Increased resistance to non-beta-lactam antibiotics was observed in the isolates harbouring both the TEM and SHV genes, of which 54% were resistant to all of the tested antibiotics except imipenem, 36% were only resistant to fluoroquinolones, and 9.1% only resistant to aminoglycosides. The possibility that the concomitant presence of TEM- and SHV-type beta-lactamases is associated with resistance to non-beta-lactam antibiotics requires further research. The prevalences of ESBL and their encoding genes in Gram-negative bacteria collected from various regions in Lebanon will now be investigated.

Inhibition of the transcription of the Escherichia coli O157:H7 genes coding for shiga-like toxins and intimin, and its potential use in the treatment of human infection with the bacterium.

Reverse-transcription PCR (RT-PCR) was used to assess the effects, in vitro, of rifampicin on the transcription of the eaeA, stx1 and stx2 genes (coding for intimin and shiga-like toxins I and II, respectively) of seven strains of Escherichia coli O157:H7 associated with human infection. Each strain was found to possess all three genes and, in the absence of rifampicin, all seven strains transcribed eaeA and stx2 (three strains did not transcribe their stx1 genes). Transcription of all three genes was inhibited (as witnessed by a negative result in the RT-PCR), however, when the strains were incubated, at 37 degrees C, with rifampicin at 4 microg/ml (found to be the minimum concentration of this antimicrobial agent that inhibited the multiplication of E. coli O157:H7). The results of an assay based on reversed passive latex agglutination (RPLA) revealed that exposure of the bacteria to 4 microg rifampicin/ml led to a 12-fold decrease in the release of shiga-like toxin I and a 16-fold decrease in the release of shiga-like toxin II. As rifampicin is capable of inhibiting the in-vitro transcription of the genes encoding the shiga-like toxins and intimin attachment protein of E. coli O157:H7, it may be useful in the treatment of human infections with strains of this bacterium. Studies are now underway to assess the in-vitro and in-vivo effects of rifampicin, at both transcription-inhibitory and bactericidal concentrations, on E. coli O157:H7. The effects of other agents on the inhibition of the expression or activity of the shiga-like toxins and intimin attachment protein will also be determined.

Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA.

We developed a PCR-based assay for the rapid and specific laboratory diagnosis of human brucellosis directly from whole blood. Specimens were collected in EDTA tubes from 17 patients with acute serologic brucellosis and 3 patients with chronic relapsing brucellosis as determined by serologic tests and the patient's clinical picture. DNA was extracted from peripheral mononuclear cells obtained from the blood of patients with brucellosis and control individuals. Specific primers for the PCR amplification of a 223-bp region on the sequence encoding the 31-kDa immunogenic Brucella abortus protein (BCSP 31) were used. All amplicons had the expected size of 223 bp. The specificity of amplification was determined by Southern hybridization and restriction endonuclease analysis. DNA extracted from blood taken from 30 healthy individuals as well as from 9 patients with typhoid fever did not show any amplification with the primers used. The test proved to be rapid and specific for the laboratory confirmation of acute human brucellosis. Further studies must be conducted to assess the utility of this test on additional patients with chronic relapsing brucellosis as well as patients under treatment.

Subtyping of Bacillus cereus by total cell protein patterns and arbitrary primer polymerase chain reaction.

Bacillus cereus is a ubiquitous sporeforming Gram-positive rod that is associated with foodborne outbreaks as well as several opportunistic infections. Inspite of the prevalence of B. cereus associated foodborne outbreaks, subtyping of the species using molecular typing assays was not attempted. In this study we have recovered 58 B. cereus isolates from natural and clinical sources and initially characterized them, along with a B. cereus strain (ATCC 14579) and B. thuringiensis natural isolate, by biotyping, antibiotic susceptibility testing, and SDS-PAGE of total cell proteins. Our data have shown the existence of 1 biotype, 3 anti-biograms and 22 (38%) total cell protein patterns among the 58 B. cereus isolates. B. thuringiensis had a different protein pattern. SDS-PAGE of total cell proteins data denote clonal heterogeneity within B. cereus. Protein pattern 4 (pp4) was the most predominant with 13 isolates of B. cereus showing this pattern. Eight out of the 13 isolates with pp4 and one B. cereus strain (ATCC 14579) were further subtyped by using the arbitrary primer polymerase chain reaction (AP-PCR) assay. Eight (88.8%) different PCR patterns out of the 9 B. cereus isolates were obtained. Patterns obtained by SDS-PAGE of total cell proteins and AP-PCR were reproducible. These results indicate that SDS-PAGE of total cell proteins allows the differentiation among species within Bacillus and of strains within B. cereus. The typability of the method was 100% and the simpson's discrimination index of diversity was 98%. The utility of SDS-PAGE of total cell proteins in a pilot epidemiologic study was assessed and results obtained demonstrate its typing potential. AP-PCR allows further subtyping of the species. Both methods if used in conjunction may be useful for further clinical and epidemiologic studies of the spectrum of diseases caused by B. cereus.

Two-step PCR-based assay for identification of bacterial etiology of otitis media with effusion in infected Lebanese children.

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella) catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was > or =2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an approximately 370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) a Haemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive for Haemophilus, 3 (8.6%) were positive for Streptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.

Detection of anti-hepatitis C-virus antibodies and hepatitis C-virus RNA in Lebanese hemodialysis patients.

Hemodialysis patients are at high risk of developing HCV infection. Reports from various countries have shown a prevalence of 12-29% among this group. The present study aimed at assessing the utility of HCV antibodies and HCV-RNA detection in the diagnosis of HCV in Lebanese hemodialysis patients. One hundred and eight hemodialysis patients from various hospitals in Lebanon were assayed for the presence of anti-HCV antibodies by ELISA and LIA, and for the presence of HCV-RNA by RT-PCR of the 5' Non-coding region (5' NCR). Specificity of the amplicons was confirmed by Southern hybridization. Seventeen out of 108 patients were reactive in ELISA and positive in the Line Immunoassay (LIA). Eleven out of the 17 were positive by RT-PCR. Three out of 29 patients nonreactive in ELISA were positive by RT-PCR. Our results indicate that hemodialysis patients in this study may be grouped into 4 categories. These include (1) patients with viremia and no immune response, (2) patients with no viremia and with an immune response, (3) patients with both viremia and immune response and (4) patients with no viremia and no immune response. The first 3 categories may reflect the different phases of HCV infection and imply that detection of both anti-HCV antibodies and HCV-RNA are needed for the establishment of adequate diagnosis. In addition, data collected from patients implicated in this study show that infection by HCV may be dialysis machine-related, rather than transfusion-related. However, cross-contamination unrelated to machines may also occurs.

Immunization of mice against Salmonella typhimurium using different DNA preparations.

Groups of female BALB/c mice were given primary and booster injections of whole genomic DNA extracted from S. typhimurium, P. aeruginosa, or S. aureus. Other groups of mice were immunized in a similar manner with the 1.57kb fragment of the mouse virulence gene (mviA), pTargeT vector (plasmid DNA)/1.57kb construct, pTargeT vector, or saline. Mice in all groups were challenged intraperitoneally with 100 LD50 of S. typhimurium. The bacterial genomic DNA was extracted using the Pure Gene extraction kit. Specific primers were used to amplify the 1.57kb fragment by PCR. The pTargeT Mammalian Expression Vector System was used to prepare the plasmid/ 1.57kb construct. Bacterial genomic DNA extracted from P. aeruginosa and S. aureus appeared to induce non-specific resistance in mice. Specific, in addition to non-specific resistance appeared to be induced when genomic DNA from S. typhimurium was used. There was a prolongation of survival in the groups of mice that received either the 1.57kb fragment or the pTargeT vector/1.57kb construct and 16.67% and 33.34% respectively, of mice in each group survived at 40 days post challenge. None of the mice in the saline control group survived by day 7 post challenge. It is suggested that the non-specific resistance observed in this study might have been due to the adjuvant effect of the non-methylated CpG and other immunostimulatory motifs in bacterial DNA. Specific resistance obtained when genomic DNA from S. typhimurium was used might have been due to minute antigenic contamination, or virulence factor genes other than the mviA gene, might have been expressed in the host, which induced specific immunity.

Genotyping of hepatitis C virus isolates from Lebanese hemodialysis patients by reverse transcription-PCR and restriction fragment length polymorphism analysis of 5' noncoding region.

We have genotyped the 5' noncoding region of hepatitis C virus in Lebanese hemodialysis patients by reverse transcription-PCR and restriction fragment length polymorphism analysis. Of 50 patients, 15 had the expected 268-bp amplicon by reverse transcription-PCR. Specificity of the amplicons was confirmed by Southern hybridization. Restriction analysis of the amplicons showed the pattern for genotype 4 (common in the Middle East).

PCR-based detection, restriction endonuclease analysis, and transcription of tonB in Haemophilus influenzae and Haemophilus parainfluenzae isolates obtained from children undergoing tonsillectomy and adenoidectomy.

We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus parainfluenzae from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the tonB gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the tonB gene was detected in both species. It is known to encode a virulent protein, TonB, in H. influenzae; however, demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that the tonB gene is apparently structurally the same in both species, with possible differences that may exist in certain H. parainfluenzae isolates. RT-PCR done on selected numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.

Comparative analysis between Pseudomonas aeruginosa genotypes and severity of symptoms in patients with unilateral or bilateral otitis externa.

Random amplified polymorphic DNA (RAPD) analysis was done on 32 isolates of Pseudomonas aeruginosa. These isolates were obtained from 22 patients who presented to the emergency room in a major medical center in Beirut, Lebanon, during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients had yellowish to greenish discharge, moderate to severe external auditory canal swelling, moderate to severe pain, and periauricular cellulitis. None of these patients had intrinsic predisposing factors. An ear swab was obtained from both ears of patients, cultured on trypticase soy agar. P. aeruginosa was identified on the basis of pyocyanine production and API identification kits. RAPD analysis was done by using two primers (10 mer and 21 mer primers) and appropriate PCR conditions on extracted DNA. Our data have shown 23 RAPD patterns (A-W) distributed among the 32 P. aeruginosa isolates. RAPD patterns were reproducible. Twenty of 32 isolates were recovered from 10 patients with bilateral otitis externa. The remaining 12 of 32 isolates were recovered from 12 different patients with unilateral otitis externa. Eleven RAPD patterns (A,B,C,D,E,F,H,I,R,U,V) were associated with severe clinical symptoms, including severe pain, severe external auditory canal swelling, periauricular cellulitis, and a yellowish discharge. The remaining RAPD patterns were not associated with severe infections. This denotes a possible association between certain genotypes and severity of symptoms.