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issn:1476-0.11
1.  Factors predicting prolonged empirical antifungal treatment in critically ill patients 
Objective
To determine the incidence, risk factors, and impact on outcome of prolonged empirical antifungal treatment in ICU patients.
Methods
Retrospective observational study performed during a one-year period. Patients who stayed in the ICU >48 h, and received empirical antifungal treatment were included. Patients with confirmed invasive fungal disease were excluded. Prolonged antifungal treatment was defined as percentage of days in the ICU with antifungals > median percentage in the whole cohort of patients.
Results
Among the 560 patients hospitalized for >48 h, 153 (27%) patients received empirical antifungal treatment and were included in this study. Fluconazole was the most frequently used antifungal (46% of study patients). Median length of ICU stay was 19 days (IQR 8, 34), median duration of antifungal treatment was 8 days (IQR 3, 16), and median percentage of days in the ICU with antifungals was 48% (IQR 25, 80). Seventy-seven patients (50%) received prolonged empirical antifungal treatment. Chemotherapy (OR [95% CI] 2.6 [1.07-6.69], p = 0.034), and suspected infection at ICU admission (3.1 [1.05-9.48], p = 0.041) were independently associated with prolonged empirical antifungal treatment.
Duration of mechanical ventilation and ICU stay were significantly shorter in patients with prolonged empirical antifungal treatment compared with those with no prolonged empirical antifungal treatment. However, ICU mortality was similar in the two groups (46 versus 52%, p = 0.62).
Conclusion
Empirical antifungal treatment was prescribed in a large proportion of study patients. Chemotherapy, and suspicion of infection at ICU admission are independently associated with prolonged empirical antifungal treatment.
doi:10.1186/1476-0711-13-11
PMCID: PMC3984712  PMID: 24621182
Antifungal treatment; Empirical treatment; Fungal infection; Invasive fungal disease; De-escalation
2.  Reviewer acknowledgement 2013 
Contributing reviewers
Annals of Clinical Microbiology and Antimicrobials would like to thank the following colleagues for their assistance with peer review of manuscripts for the journal in 2013.
doi:10.1186/1476-0711-13-8
PMCID: PMC3931273
3.  Prevalence of intestinal parasite, Shigella and Salmonella species among diarrheal children in Jimma health center, Jimma southwest Ethiopia: a cross sectional study 
Background
Diarrheal disease continues to be an important cause of morbidity and mortality among young children in developing countries including Ethiopia. Globally, intestinal parasite, Shigella and Salmonella species remain major contributors to acute enteric infections. The study was aimed at determining the frequency of intestinal parasite, Shigella and Salmonella species identified from diarrheic children at Jimma Health Centre, Jimma south west Ethiopia.
Methods
A health institution based cross sectional study was conducted from March to November 2012. A structured questionnaire was used for collection of data on socio- demographic characteristics. Parasite and bacteria identification as well as susceptibility testing was done using standard parasitological and bacteriological procedures.
Results
A total of 260 diarrheal children were included in the study. A total of 129 (49.6%) samples were positive for intestinal parasite, Shigella and Salmonella species. Of these, 107 (41.1%), 6 (2.3%) and 16 (6.2%) samples were positive for intestinal parasite, Shigella and Salmonella species respectively. The dominant isolated parasite was G. lamblia with prevalence of 13.5% followed by A. lumbricoides (11.5%). The least identified parasites were Schistosoma mansoni and Taenia species accounting 0.4% each. Multiple parasitic infections were observed in 19 (7.3%) patients. Shigella species showed hundred percent resistances to ampicillin, amoxacillin, and cotrimoxazole. All Salmonella isolates were resistant against amoxicillin. All Shigella and Salmonella species were susceptible to ceftriaxone, ciprofloxacin and gentamycin.
Conclusion
The presence of reasonably high amount of intestinal parasite and Salmonella and Shigella species that are drug resistance to the commonly prescribed drugs is a treat to the children and community at large. Therefore, measures including health education, improvement of safe water supply, sanitation facilities and continuous monitoring of microbiological and antimicrobial surveillance is crucial.
doi:10.1186/1476-0711-13-10
PMCID: PMC3922032  PMID: 24499189
Intestinal parasite; Shigella; Salmonella; Susceptibility test; Jimma; Ethiopia
4.  Sacroiliitis secondary to catheter-related bacteremia due to Mycobacterium abscessus (sensu stricto) 
We describe a case of sacroiliitis secondary to catheter-related bacteremia due to Mycobacterium abscessus (sensu stricto). This case confirms that MultiLocus sequence typing and variable-number tandem-repeat methods are very robust techniques to identify the pathogen species and to validate molecular epidemiological links among complex M. abscessus isolates.
doi:10.1186/1476-0711-13-9
PMCID: PMC3943385  PMID: 24479655
Sacroiliitis; Catheter-related; Bacteremia; Mycobacterium abscessus complex; MultiLocus sequence typing method; Variable-number tandem-repeat method
5.  Effects of Carbapenem consumption on the prevalence of Acinetobacter infection in intensive care unit patients 
Background
The consumption of carbapenems has increased worldwide, together with the increase in resistant gram negative bacilli. Subsequently, the prevalence of carbapenem-resistant Acinetobacter infections has increased rapidly and become a significant problem particularly in intensive care unit patients. The aim of the present study was to evaluate the changes in the prevalence of Acinetobacter infection by restricting the consumption of carbapenems in intensive care unit patients.
Methods
This study was conducted between May 1, 2011 and February 28, 2013. The amount of carbapenem consumption and the number of patients with multi-drug resistant Acinetobacter baumannii (MDRAB) isolates during the study period were retrospectively obtained from the records of the patients, who were hospitalized in the intensive care unit. The study period was divided into two periods named as: Carbapenem non-restricted period (CNRP) and carbapenem-restricted period (CRP). During CNRP, no restrictions were made on the use of carbapenems. During CRP, the use of carbapenems was not allowed if there was an alternative to carbapenems. Primary Endpoint: MDRAB infection after ICU admission. The definition of nosocomial infections related to Acinetobacter spp. was based on the criteria of the Center for Disease Control (CDC). The correlation between the amount of carbapenem consumption and the number of infections with MDRAB strains between the two periods were evaluated.
Results
During the study period, a total of 1822 patients’ (1053 patients in CNRP and 769 patients in CRP) records were evaluated retrospectively. A total of 10.82 defined daily dose (DDD/100 ICU days) of anti-pseudomonal carbapenem were used in CNRP, and this figure decreased to 6.95 DDD/100 ICU days in CRP. In the 8-month CNRP, 42 (3.98%) MDRAB-related nosocomial infections were detected, and 14 (1.82%) infections were detected in CRP (p = 0.012).
Conclusion
The prevalence of MDRAB strains isolated in the CNRP was 2.24-fold higher than the prevalence in the CRP. The prevalence of Acinetobacter infections can be reduced by taking strict isolation measures as well as by implementing good antibiotics usage policy.
doi:10.1186/1476-0711-13-7
PMCID: PMC3898784  PMID: 24405720
Carbapenem; Acinetobacter infection; Carbapenem consumption
6.  Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico 
Background
Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors.
Methods
We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE).
Results
In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination.
Conclusion
These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections.
doi:10.1186/1476-0711-13-6
PMCID: PMC3905915  PMID: 24405688
MRSA; Haemodialysis catheter; Virulence factors
7.  The interrelations of radiologic findings and mechanical ventilation in community acquired pneumonia patients admitted to the intensive care unit: a multicentre retrospective study 
Background
We evaluated patients admitted to the intensive care units with the diagnosis of community acquired pneumonia (CAP) regarding initial radiographic findings.
Methods
A multicenter retrospective study was held. Chest x ray (CXR) and computerized tomography (CT) findings and also their associations with the need of ventilator support were evaluated.
Results
A total of 388 patients were enrolled. Consolidation was the main finding on CXR (89%) and CT (80%) examinations. Of all, 45% had multi-lobar involvement. Bilateral involvement was found in 40% and 44% on CXR and CT respectively. Abscesses and cavitations were rarely found. The highest correlation between CT and CXR findings was observed for interstitial involvement. More than 80% of patients needed ventilator support. Noninvasive mechanical ventilation (NIV) requirement was seen to be more common in those with multi-lobar involvement on CXR as 2.4-fold and consolidation on CT as 47-fold compared with those who do not have these findings. Invasive mechanical ventilation (IMV) need increased 8-fold in patients with multi-lobar involvement on CT.
Conclusion
CXR and CT findings correlate up to a limit in terms of interstitial involvement but not in high percentages in other findings. CAP patients who are admitted to the ICU are severe cases frequently requiring ventilator support. Initial CT and CXR findings may indicate the need for ventilator support, but the assumed ongoing real practice is important and the value of radiologic evaluation beyond clinical findings to predict the mechanical ventilation need is subject for further evaluation with large patient series.
doi:10.1186/1476-0711-13-5
PMCID: PMC3898785  PMID: 24400646
Radiography; Thoracic; Pneumoniae; Imaging; Critical care
8.  Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131 
Background
The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.
Methods
A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.
Results
Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The blaCTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The blaCTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.
Conclusions
Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.
doi:10.1186/1476-0711-13-4
PMCID: PMC3898780  PMID: 24397567
β-lactam resistance; Class A β-lactamases; PFGE; ST131; Saudi Arabia
9.  Real-time PCR TaqMan assay for rapid screening of bloodstream infection 
Background
Sepsis is one of the main causes of mortality and morbidity. The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and better prognosis. This study aimed to accelerate the detection and discrimination of Gram-positive (GP) and Gram-negative (GN) bacteria and Candida species in blood culture samples by molecular methods.
Methods
The Real-GP®, -GN®, and -CAN® real-time PCR kit (M&D, Wonju, Republic of Korea) assays use the TaqMan probes for detecting pan-GP, pan-GN, and pan-Candida species, respectively. The diagnostic performances of the real-time PCR kits were evaluated with 115 clinical isolates, 256 positive and 200 negative blood culture bottle samples, and the data were compared to results obtained from conventional blood culture.
Results
Eighty-seven reference strains and 115 clinical isolates were correctly identified with specific probes corresponding to GP-bacteria, GN-bacteria and Candida, respectively. The overall sensitivity and specificity of the real-time PCR kit with blood culture samples were 99.6% and 89.5%, respectively.
Conclusions
The Real-GP®, -GN®, and -CAN® real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs).
doi:10.1186/1476-0711-13-3
PMCID: PMC3898783  PMID: 24393579
Real-time polymerase chain reaction; Blood culture; Gram-positive bacteria; Gram-negative bacteria; Candida
10.  Molecular identification of clinical “difficult-to-identify” microbes from sequencing 16S ribosomal DNA and internal transcribed spacer 2 
Background
Clinical microbiology laboratories have to accurately identify clinical microbes. However, some isolates are difficult to identify by the automated biochemical text platforms, which are called “difficult-to-identify” microbes in this study. Therefore, the ability of 16S ribosomal DNA (16S rDNA) and internal transcribed spacer 2 (ITS2) sequencing to identify these “difficult-to-identify” bacteria and fungi was assessed in this study.
Methods
Samples obtained from a teaching hospital over the past three years were examined. The 16S rDNA of four standard strains, 18 clinical common isolates, and 47 “difficult-to-identify” clinical bacteria were amplified by PCR and sequenced. The ITS2 of eight standard strains and 31 “difficult-to-identify” clinical fungi were also amplified by PCR and sequenced. The sequences of 16S rDNA and ITS2 were compared to reference data available in GenBank by using the BLASTN program. These microbes were identified according to the percentage of similarity to reference sequences of strains in GenBank.
Results
The results from molecular sequencing methods correlated well with automated microbiological identification systems for common clinical isolates. Sequencing results of the standard strains were consistent with their known phenotype. Overall, 47 “difficult-to-identify” clinical bacteria were identified as 35 genera or species by sequence analysis (with 10 of these identified isolates first reported in clinical specimens in China and two first identified in the international literature). 31 “difficult-to-identify” clinical fungi tested could be identified as 15 genera or species by sequence analysis (with two of these first reported in China).
Conclusions
Our results show the importance of 16S rDNA and internal ITS2 sequencing for the molecular identification of “difficult-to-identify” bacteria and fungi. The development of this method with advantages of convenience, availability, and cost-effectiveness will make it worth extending into clinical practice in developing countries.
doi:10.1186/1476-0711-13-1
PMCID: PMC3905965  PMID: 24383440
Bacteria; Fungi; 16S rDNA; Internal transcribed spacer 2
11.  Prevalence of antibiotic resistance in multi-drug resistant coagulase-negative staphylococci isolated from invasive infection in very low birth weight neonates in two Polish NICUs 
Background
Multi-drug resistant coagulaso-negative staphylococci (CNS) have become an increasing problem in nosocomial infections connected with the presence of medical devices. The paper aimed to analyze the prevalence of antibiotic resistance in CNS isolated from invasive infection in very low birth weight (VLBW) neonates.
Methods
Continuous prospective target surveillance of infections was conducted in 2009 at two Polish NICUs that participated in the Polish Neonatology Surveillance Network (PNSN). The study covered 386 neonates with VLBW (≤1500 g), among which 262 cases of invasive infection were detected with predominance of CNS (123; 47%). Altogether, 100 CNS strains were analyzed. The resistance phenotypes were determined according to EUCAST. Resistance genes: mecA, ermA, ermB, ermC, msrA, aac(6')/aph(2''), ant(4')-Ia and aph(3')-IIIa were detected using multiplex PCR.
Results
The most common species was S. epidermidis (63%), then S. haemolyticus (28%) and other CNS (9%). Among S. epidermidis, 98% of isolates were resistant to methicillin, 90% to erythromycin, 39% to clindamycin, 95% to gentamicin, 60% to amikacin, 36% to ofloxacin, 2% to tigecycline, 3% to linezolid and 13% to teicoplanin. Among S. haemolyticus isolates, 100% were resistant to methicillin, erythromycin and gentamicin, 18% to clindamycin, 50% to amikacin, 86% to ofloxacin, 14% to tigecycline and 4% to teicoplanin. No resistance to linezolid was detected for S. haemolyticus isolates. Moreover, all isolates of S. epidermidis and S. haemolyticus were susceptible to vancomycin. The mecA gene was detected in 98% of S. epidermidis isolates and all of S. haemolyticus ones. Among macrolide resistance isolates, the ermC was most common in S. epidermidis (60%) while msrA was prevalent in S. haemolyticus (93%). The ermC gene was indicated in all isolates with cMLSB, whereas mrsA was found in isolates with MSB phenotype. Of the aminoglycoside resistance genes, aac(6')/aph(2'') were present alone in 83% of S. epidermidis, whereas aac(6')/aph(2'') with aph(3')-IIIa were predominant in 84% of S. haemolyticus.
Conclusions
Knowing the epidemiology and antibiotic resistance of CNS isolated from invasive infection in VLBW neonates is a key step in developing targeted prevention strategies and reducing antibiotic consumption.
doi:10.1186/1476-0711-12-41
PMCID: PMC3898809  PMID: 24359473
Multi-drug resistant coagulase-negative staphylococci; Resistance genes; Very-low-birth-weight neonates; Nosocomial infections
12.  Recurrent cryptococcal immune reconstitution inflammatory syndrome in an HIV-infected patient after anti-retroviral therapy: a case report 
Cryptococcal immune reconstitution inflammatory syndrome (C-IRIS) in HIV-infected patients presents as a clinical worsening or new presentation of cryptococcal disease as a result of anti-retroviral therapy mediated immune restoration. Recurrent C-IRIS is a rare condition. Recently, recurrent C-IRIS involving the central nervous system, which is thought to require prolonged or alternative immunosuppressive therapy, has been described. Here, we present an unusual case of recurrent C-IRIS, sequentially involving the central nervous system and lymph nodes, in an HIV-infected patient after anti-retroviral therapy. While corticosteroids were used to control the inflammatory cerebral cryptococcomas, lymphadenitis that developed after cessation of corticosteroids resolved without additional immunosuppressive or anti-inflammatory drugs. This case suggests the possibility of site-specific recovery of pathogen-specific immune response after anti-retroviral therapy. In this condition, each episode of C-IRIS may be treated independently, and extended corticosteroids may not always be needed.
doi:10.1186/1476-0711-12-40
PMCID: PMC3909341  PMID: 24354779
Cryptococcal; Recurrent; Immune reconstitution inflammatory syndrome; Lymphadenitis
13.  Bacterial contamination, bacterial profile and antimicrobial susceptibility pattern of isolates from stethoscopes at Jimma University Specialized Hospital 
Introduction
Hospital acquired infections are recognized as critical public health problems. Infections are frequently caused by organisms residing in healthcare environment, including contaminated medical equipment like Stethoscopes.
Objective
To determine bacterial contamination, bacterial profile and anti-microbial susceptibility pattern of the isolates from stethoscopes at Jimma University Specialized Hospital.
Methodology
Cross-sectional study conducted from May to September 2011 at Jimma University Specialized Hospital. One hundred seventy-six stethoscopes owned by Health Care Workers (HCWs) and Medical students were randomly selected and studied. Self-administered structured questionnaire was used to collect socio-demographic data. Specimen was collected using moisten sterile cotton swab and 1 ml normal saline was used to transport the specimen, all laboratory investigations were done following standard microbiological techniques, at Microbiology Laboratory, Jimma University. SPSS windows version 16 used for data analysis and P <0.05 was considered statistically significant. Result: A total, of 151 (85.8%) stethoscopes were contaminated. A total of 256 bacterial strains and a mean of 1.44×104 CFUs/diaphragm of stethoscopes was isolated. Of the 256 isolates, 133 (52%) were potential pathogens like S. aureus, Klebsiella spp., Citrobacter spp., Salmonella spp., Proteus spp., Enterobacter spp., P. aeruginosa and E. coli. All strains were resistant to multiple classes of antibiotics (two to eight classes of antibiotics). Disinfection practice was poor. Disinfection practice was found to be associated with bacterial contamination of stethoscopes (P < 0.05). High contamination rate 100 (90.9%) was observed among stethoscopes that had never been disinfected; while the least contamination 29 (72.2%) was found on those disinfected a week or less before the survey.
Conclusion
Bacterial contamination of the stethoscope was significant. The isolates were potential pathogens and resistant to multiple classes of antibiotics. Stethoscope is potential vehicle in the transmission of infections between patients and Healthcare Workers. Stethoscope diaphragm should be disinfected before and after each patient contact.
doi:10.1186/1476-0711-12-39
PMCID: PMC3880102  PMID: 24330702
14.  Colistin use in pediatric intensive care unit for severe nosocomial infections: experience of an university hospital 
Background
The aim of this study was to investigate the efficacy and safety of colistin therapy in pediatric patients with severe nosocomial infections in pediatric intensive care unit.
Methods
The medical records of patients treated with colistin at a 200-bed university children hospital were reviewed.
Result
Thirty-one patients (male/female = 22/9; median age, 3 years; range, 3 months-17 years) received forty-one courses of colistin. The average dose of colistin was 4.9 ± 0.5 mg/kg/day and average treatment duration was 19.8 ± 10.3 days. Three patients who received concomitant nephrotoxic agent with colistin developed nephrotoxicity. Colistin treatment was well tolerated in other patients, and neurotoxicity was not seen in any patient. Favourable outcome was achieved in 28 (68.3%) episodes. Twelve patients died during the colistin therapy. Six of these patients died because of primary underlying disease. The infection-related mortality rate was found 14.6% in this study.
Conclusion
In our study, colistin therapy was found to be acceptable treatment option for the severe pediatric nosocomial infections caused by multi-drug resistant bacteria. However, the use of concomitant nephrotoxic drugs with colistin must be avoided and renal function test should be closely monitored.
doi:10.1186/1476-0711-12-32
PMCID: PMC3827824  PMID: 24199612
Colistin; Child; Multi-drug resistant bacteria; Nosocomial infection; Nephrotoxicity
15.  The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in bacteria 
The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of blaNDM and blaKPC with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for blaNDM (R2 = 0.971; slope, -3.273) and blaKPC (R2 = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.
doi:10.1186/1476-0711-12-30
PMCID: PMC3816589  PMID: 24143953
Duplex; Real-time PCR; Infection control; Carbapenemase
16.  Analysis of integrons and associated gene cassettes in clinical isolates of multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria 
Background
Multidrug resistant Pseudomonas aeruginosa harbours integrons and other mobile genetic elements such as plasmids and transposons, which easily disseminate antibiotic resistance genes among clinical strains of P. aeruginosa.
Methodology
Plasmid extraction of 54 clinical isolates of P. aeruginosa was carried out by alkaline lysis method; and plasmid size estimation was done by using E. coli V517 standard plasmid marker. Fifty-four clinical strains of P. aeruginosa were isolated from 5 hospitals in 3 Southwestern states of Nigeria between March and September 2010. Plasmid extraction of isolates was carried out by alkaline lysis method; and plasmid size estimation was done by using E. coli V517 standard plasmid marker. PCR amplification for the 3 classes of resistance integrons, and gene cassette characterization were carried out using specific primers and by sequencing of PCR products. Conjugal mating of the integron positive P. aeruginosa strains with E. coli DH5α was performed to demonstrate transferability of integrons and gene cassettes.
Result
Agarose gel electrophoresis of plasmid DNA revealed that all the 54 P. aeruginosa harboured 1–4 plasmids with sizes ranging from 2.2 – >58 kb. Class 1 integron was identified in 31 (57%) strains; but none of them carried class 2 and class 3 integrons. High prevalence of aadA gene conferring resistance to streptomycin/spectinomycin was detected in the strains positive for class 1 integron. Sequencing of the 1.6 kb and 1.2 kb amplified band of gene cassettes revealed the presence of aadA6-orfD and aadA13 respectively.
Conclusion
This study demonstrates the presence of plasmids and integrons harbouring resistance gene cassettes, which may collectively constitute an efficient system for dissemination of resistance genes in P. aeruginosa. Disturbingly, the rapid and unabated spread of class 1 integron-associated multidrug resistant P. aeruginosa in Southwest Nigeria may greatly hamper successful treatment of infections caused by such strains. This necessitates the establishment of functional antimicrobial resistance surveillance programmes in Nigeria.
doi:10.1186/1476-0711-12-29
PMCID: PMC3842740  PMID: 24143920
Pseudomonas aeruginosa; Antibiotic resistance; Plasmids; Integrons; Gene cassettes
17.  Antimicrobial resistance in human and animal pathogens in Zambia, Democratic Republic of Congo, Mozambique and Tanzania: an urgent need of a sustainable surveillance system 
A review of the published and unpublished literature on bacterial resistance in human and animals was performed. Sixty-eight articles/reports from the Democratic Republic of Congo (DRC), Mozambique, Tanzania and Zambia were reviewed. The majority of these articles were from Tanzania. There is an increasing trend in the incidence of antibiotic resistance; of major concern is the increase in multidrug- resistant Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholera, non-typhoid Salmonella and other pathogens responsible for nosocomial infections. The increase in methicillin- resistant Staphylococcus aureus and extended-spectrum beta-lactamase (ESBL) producers in the countries under review confirms the spread of these clones worldwide. Clinical microbiology services in these countries need to be strengthened in order to allow a coordinated surveillance for antimicrobial resistance and provide data for local treatment guidelines and for national policies to control antimicrobial resistance. While the present study does not provide conclusive evidence to associate the increasing trend in antibiotic resistance in humans with the use of antibiotics in animals, either as feed additives or veterinary prescription, we strongly recommend a one-health approach of systematic surveillance across the public and animal health sectors, as well as the adherence to the FAO (Food and Agriculture Organization)-OIE (World Organization of animal Health) –WHO(World Health Organization) recommendations for non-human antimicrobial usage.
doi:10.1186/1476-0711-12-28
PMCID: PMC3852305  PMID: 24119299
18.  Antibacterial activity of Nymphaea nouchali (Burm. f) flower 
Background
The present work aimed to find out the antibacterial activity of Nymphaea nouchali flower on human and plant pathogenic bacteria.
Methods
Antibacterial potency of methanol, acetone, ethyl acetate and petroleum spirit extracts of Nymphaea nouchali flower has been tested against four human pathogenic bacteria Bacillus subtilis (FO 3026) Escherichia coli (IFO 3007), Klebsiella pneumonia (ATTC 10031) and Sarcina lutea (IFO 3232) and one plant pathogenic bacterium Xanthomonas campestris (IAM 1671) by disc diffusion assay. Zone of inhibition produced by different extracts against the test bacteria was measured and compared with standard antibiotic disc.
Results
Methanol extract possessed better antibacterial activity against two pathogenic bacteria, B. subtilis (FO 3026) and S. lutea (IFO 3232) than commercial antibiotic nalidixic acid. Acetone extract showed moderate sensitivity whereas B. subtilis (FO 3026), S. lutea (IFO 3232) and X. campestris (IAM 1671) showed resistance to ethyl acetate and petroleum spirit extracts. The minimum inhibitory concentrations of various extracts were ranged between 128–2048 μgml-1.
Conclusions
Nymphaea nouchali flower could be a potential candidate for future development of novel broad spectrum antibacterial herbal formulation.
doi:10.1186/1476-0711-12-27
PMCID: PMC3852100  PMID: 24099586
Nymphaea nouchali flower; Antibacterial activity; Disc diffusion assay; Nalidixic acid
19.  In vitro antibacterial activity of Tabernaemontana alternifolia (Roxb) stem bark aqueous extracts against clinical isolates of methicillin resistant Staphylococcus aureus 
Background
The rise of antibiotic resistance among methicillin resistant Staphylococcus aureus (MRSA), have caused concerns for the treatment of MRSA infections. Hence, search for an alternative therapy for these infections is inevitable. Folk Indian medicine refers to the use of leaf and stem bark powder of Tabernaemontana alternifolia (Roxb) in treatment of skin infections, but no scientific report establishes its antibacterial activity.
Methods
Direct aqueous extracts and sequential aqueous extracts of the stem bark of T. alternifolia (using petroleum ether and ethyl acetate as other solvents) were prepared by soxhlet extraction. The antibiotic sensitivity profiles of the clinical isolates were determined against 18 antibiotics using disc diffusion method. The isolates were identified by 16S rRNA gene sequencing. The methicillin resistance among S. aureus (MRSA) was confirmed by PCR amplification of mecA gene. The disc diffusion method was used to determine the antibacterial activity of the extracts. The micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the extract against the test organism. To further evaluate the therapeutic potential of the extract, cell cytotoxicity was checked on Vero cells by MTT assay. Chemical profiling of the extract was done by HPTLC method.
Results
The aqueous extracts of T. alternifolia stem bark exhibited antibacterial activity against Gram-positive microorganisms, particularly against clinical isolates of MRSA and vancomycin resistant S. aureus (VRSA). The minimum inhibitory concentration (MIC) of extract against the isolates ranged from 600–800 μg/ml. The extract did not exhibit cytotoxic activity against Vero cells even at the concentration of 4 mg/ml. The chemical profiling revealed presence of alkaloids, flavonoids, coumarins, saponins and steroids. Petroleum ether and ethyl acetate extracts did not exhibit antibacterial activity.
Conclusion
Our results offer a scientific basis for the traditional use of T. alternifolia in the treatment of skin infections, showing that the plant extract has an enormous potential as a prospective alternative therapy against MRSA skin infections. The present study lays the basis for future studies, to validate the possible use of T. alternifolia as a candidate in the treatment of MRSA infections.
doi:10.1186/1476-0711-12-26
PMCID: PMC3851168  PMID: 24066905
Tabernaemontana alternifolia (Roxb); Anti-MRSA; Cytotoxicity; Plant extract; Antimicrobial
20.  Increased serum procalcitonin levels in pregnant patients with asymptomatic bacteriuria 
Background
Among the pregnancy urinary tract infections, asymptomatic bacteriuria (ASB) is the most common one. Untreated ASB can progress to pyelonephritis in 30-50% of the patients and can also result in prematurity in 27% of the pregnancy so it needs immediate diagnosis and treatment. In this study, we wanted to evaluate procalcitonin levels, compared to other inflammatory in pregnant women with ASB.
Methods
The study was designed between the period of January 2012 and February 2013 at Sakarya University School of Medicine, Department of Gynecology and Obstetrics. The study population included 30 pregnant patients with asymptomatic bacteriuria and 39 healthy pregnant controls.
Results
Mean age was 28 (SD, 5.5) of the study population; mean maternal weight was 70 (SD, 8) kilogram. There were no statically significant differences between the groups according to the routine biochemical parameters, but gestational age was significantly lower in the ASB group compared to the controls (20.4 vs 28.6, respectively; p < 0.001). Serum procalcitonin levels were negative in all of the controls. In ASB group, 9 (30%) patients had procalcitonin levels greater than >0.05 ng/ml and 21(70%) patients had negative procalcitonin levels (Chi-squrae, p < 0.001). The sensitivity and specificity of procalcitonin assay for ASB was calculated as 30% and 100%, respectively. The positive predictive value was 100% and the negative predictive value was 65%. The most frequent microorganisms in the urine culture were Escherichia coli (26 patients, 87%), Proteus mirabilis (3 patients, 10%) and Klebsiella (1 patient, 3%) in the ASB group. We experienced four (44%) recurrences among nine positive procalcitonin in ASB patients after completion of treatment of the first ASB diagnosis.
Discussion
Procalcitonin levels were significantly higher in ASB group than the control group and serum procalcitonin levels were higher in pregnant women with recurrent ASB. This finding is an important result revealed that high procalcitonin level can predict the further urinary tract infection risk. Finally, serum procalcitonin levels were normal in healthy pregnant women while other inflammatory markers such as WBC, ESR and CRP levels were higher.
doi:10.1186/1476-0711-12-25
PMCID: PMC3846744  PMID: 24006912
Asymptomatic bacteriuria; Procalcitonin; Pregnant women; Inflammatory markers
21.  Antimicrobial susceptibility among gram-negative isolates collected in the USA between 2005 and 2011 as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) 
Background
The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) was designed to monitor in vitro antimicrobial susceptibility to tigecycline and comparator agents. We present susceptibility data on Gram-negative organisms collected between 2005 and 2011 from nine United States census regions.
Methods
T.E.S.T. was conducted using standardized CLSI methodologies or FDA-approved breakpoints.
Results
Tigecycline was highly active (MIC90 ≤ 2 mg/L) against Enterobacteriaceae irrespective of species or region of collection (N = 25011). The isolates were also highly susceptible to the carbapenems when all regional data are combined, except for ESBL-producing Klebsiella pneumoniae (MIC90 16 mg/L) and Acinetobacter baumannii (MIC90 ≥ 32 mg/L). In addition, 883 (30%) of 2900 A. baumannii isolates were classified as multidrug-resistant (MDR): these MDR organisms were most susceptible to tigecycline (MIC90 2 mg/L) and minocycline (MIC90 8 mg/L) when all regional data are considered together. Susceptibility patterns also varied widely among the regions
Conclusions
The findings highlight the importance of monitoring antimicrobial susceptibility patterns and implementing effective methods to curb increased resistance and also confirm that additional studies to determine the efficacy of tigecycline in vivo, especially for treating infections with MDR organisms, are warranted.
doi:10.1186/1476-0711-12-24
PMCID: PMC3851274  PMID: 24006892
Surveillance; Tigecycline; Resistance; USA; Census regions
22.  Application of ATC/DDD methodology to eveluate of antibiotic use in a general hospital in Turkey 
Background
The aim of this study is to evaluate in-house antibiotic use in a state hospital in Turkey with its cost, using the ATC/DDD index, which is an accepted standard method.
Methods
This study was performed as a point prevalence study in a state hospital with 372 beds. All in-house patients using antibiotics on July 19, 2011 were included in the study. Indications for antibiotic use and information about the patients were recorded on special forms. Antibiotic use and cost analysis were evaluated using the ATC/DDD index, which is also suggested by the WHO to be used in similar studies.
Findings
147 patients out of 308 patients who were in-house were identified to use antibiotics with appropriate indications for prophylaxis or treatment in 61% of the patients. The rate of appropriate antibiotic use was identified to be in 78%, while this rate was 38.9% in surgical clinics. The daily cost of the antibiotics consumed on the date of the study was calculated as 4104.79 TL (=2476.80 USD).
Discussion
The rate of inappropriate use of antibiotics seems to be high in our hospital. This will result in both increased costs and also increased nosocomial infection rates with resistant species. Infectious disease specialists should take more active roles in the in-house antibiotic use, hospitals should prepare and implement their own principles of antibiotic use, and microbiology laboratories should be used more effectively. These measures would decrease the conspicuous shortcomings in the antibiotic use.
doi:10.1186/1476-0711-12-23
PMCID: PMC3847134  PMID: 24004538
Antibiotic; ATC/DDD index; Appropriate use of antibiotics
23.  New antibiotics for bad bugs: where are we? 
Bacterial resistance to antibiotics is growing up day by day in both community and hospital setting, with a significant impact on the mortality and morbidity rates and the financial burden that is associated. In the last two decades multi drug resistant microorganisms (both hospital- and community-acquired) challenged the scientific groups into developing new antimicrobial compounds that can provide safety in use according to the new regulation, good efficacy patterns, and low resistance profile. In this review we made an evaluation of present data regarding the new classes and the new molecules from already existing classes of antibiotics and the ongoing trends in antimicrobial development. Infectious Diseases Society of America (IDSA) supported a proGram, called “the ′10 × ´20′ initiative”, to develop ten new systemic antibacterial drugs within 2020. The microorganisms mainly involved in the resistance process, so called the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) were the main targets. In the era of antimicrobial resistance the new antimicrobial agents like fifth generation cephalosporins, carbapenems, monobactams, β-lactamases inhibitors, aminoglycosides, quinolones, oxazolidones, glycopeptides, and tetracyclines active against Gram-positive pathogens, like vancomycin-resistant S. aureus (VRSA) and MRSA, penicillin-resistant streptococci, and vancomycin resistant Enterococcus (VRE) but also against highly resistant Gram-negative organisms are more than welcome. Of these compounds some are already approved by official agencies, some are still in study, but the need of new antibiotics still does not cover the increasing prevalence of antibiotic-resistant bacterial infections. Therefore the management of antimicrobial resistance should also include fostering coordinated actions by all stakeholders, creating policy guidance, support for surveillance and technical assistance.
doi:10.1186/1476-0711-12-22
PMCID: PMC3846448  PMID: 23984642
New antibiotics; Resistance; Bacteria; FDA; EMA
24.  Molecular epidemiological study of clinical Acinetobacter baumannii isolates: phenotype switching of antibiotic resistance 
Background
The presence of clinical Acinetobacter baumannii (A. baumannii) isolates with differing antibiotic resistance phenotypes in the same patient causes difficulties and confusion in treatment. This phenomenon may be caused by reasons such as cross-infection from neighboring patients that switches to different A. baumannii strain, natural mutation of A. baumannii, inducing of different antibiotic resistance genes expression or acquisition of genes conferring resistance from another source. To elucidate this question, clinical A. baumannii strains, isolated from the same individual patients, showed antibiotic resistance phenotypes switching during the same hospitalization period, were attentively collected for further analysis. Molecular approaches for phylogenetic analysis, including pulsed field gel electrophoresis, multilocus sequence typing, and short tandem repeat analysis, were employed for the chronological studies.
Findings
Our results showed that antibiotic resistance phenotype switching could have occurred as a result through both cross-infection and natural mutation roots. Our results also suggest that rapid phenotype switching between paired isolates could occur during one single course of antibiotic treatment.
Conclusions
Though cross infection caused antibiotic resistance phenotype switching does occur, natural mutation of A. baumannii isolates is particularly cautious for antibiotic treatment.
doi:10.1186/1476-0711-12-21
PMCID: PMC3851446  PMID: 23965155
Acinetobacter baumannii; Pulsed field gel electrophoresis; Multilocus sequence typing; Short tandem repeat; Phenotype switch
25.  Antibacterial activity of Aquilaria crassna leaf extract against Staphylococcus epidermidis by disruption of cell wall 
Background
Aquilaria crassna Pierre ex Lecomte has been traditionally used in Thailand for treatment of infectious diseases such as diarrhoea and skin diseases for a long time. The main objectives of this study were to examine antibacterial activity of the Aquilaria crassna leaf extract against Staphylococcus epidermidis and its underlying mechanism. The antioxidant activity and acute toxicity were studied as well.
Methods
Antioxidant activities were examined by FRAP, ABTS and DPPH scavenging methods. Antibacterial activity was conducted using disc diffusion assay and the minimum inhibitory concentration (MIC) was determined by dilution method. The minimum bactericidal concentration (MBC) was reported as the lowest concentration producing no growth of microbes in the subcultures. Morphological changes of the microbe were observed by scanning electron microscopy, while an inhibitory effect on biofilm formation was evaluated by phase contrast microscopic analysis. Bacterial cell wall integrity was assessed by transmission electron microscopy. Acute toxicity was conducted in accordance with the OECD for Testing of Chemicals (2001) guidelines.
Results
The extract exhibited considerable antioxidant activity. Staphylococcus epidermidis was susceptible to the extract with the MIC and MBC of 6 and 12 mg/ml, respectively. The extract caused swelling and distortion of bacterial cells and inhibited bacterial biofilm formation. Rupture of bacterial cell wall occurred after treated with the extract for 24 h. Acute toxicity test in mice showed no sign of toxicity or death at the doses of 2,000 and 15,000 mg/kg body weight.
Conclusion
The aqueous extract of Aquilaria crassna leaves possesses an in vitro antibacterial activity against Staphylococcus epidermidis, with no sign of acute oral toxicity in mice, probably by interfering with bacterial cell wall synthesis and inhibiting biofilm formation.
doi:10.1186/1476-0711-12-20
PMCID: PMC3765429  PMID: 23962360
Aquilaria crassna; Staphylococcus epidermidis; Antioxidant; Anitbacterials; Cell wall; Acute toxicity

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