Extended spectrum beta-lactamase and metallo beta-lactamase production among <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from different clinical samples in a tertiary care hospital in Kathmandu,Nepal

Background

Extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production in Klebsiella pneumoniae and Escherichia coli are the commonest modes of drug resistance among these commonly isolated bacteria from clinical specimens. So the main purpose of our study was to determine the burden of ESBL and MBL production in E. coli and K. pneumoniae isolated from clinical samples. Further, the antimicrobial susceptibility patterns of E. coli and K. pneumoniae were also determined.

Methods

A cross-sectional study was conducted at Om Hospital and Research Centre, Kathmandu, Nepal by using the E. coli and K. pneumoniae isolated from different clinical samples (urine, pus, body fluids, sputum, blood) from May 2015 to December 2015. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Extended spectrum beta-lactamase production was detected by combined disc method using ceftazidime and ceftazidime/clavulanic acid discs and cefotaxime and cefotaxime/clavulanic acid discs. Similarly, metallo beta-lactamase production was detected by combined disc assay using imipenem and imipenem/ethylenediaminetetracetate discs. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug resistant (MDR).

Results

Of total 1568 different clinical samples processed, 268 (17.1%) samples were culture positive. Among which, E. coli and K. pneumoniae were isolated from 138 (51.5%) and 39 (14.6%) samples respectively. Of the total isolates 61 (34.5%) were ESBL producers and 7 (4%) isolates were found to be MBL producers. High rates of ESBL production (35.9%) was noted among the clinical isolates from outpatients, however no MBL producing strains were isolated from outpatients. Among 138 E. coli and 39 K. pneumoniae, 73 (52.9%) E. coli and 23 (59%) K. pneumoniae were multidrug resistant. The lowest rates of resistance was seen toward imipenem followed by piperacillin/tazobactam, amikacin and cefoperazone/sulbactam.

Conclusions

High rate of ESBL production was found in the E. coli and K. pneumoniae isolated from outpatients suggesting the dissemination of ESBL producing isolates in community. This is very serious issue and can’t be neglected. Regular monitoring of rates of ESBL and MBL production along with multidrug resistance among clinical isolates is very necessary.

     

Antimicrobial misuse in pediatric urinary tract infections: recurrences and renal scarring

Background

In children, urinary tract infection (UTI) is one of a common bacterial infection. This study was conducted to detect the uropathogen, antimicrobial susceptibility, pathogen associated with recurrences and renal scarring in children initially taken care from general practitioners and later presented to tertiary care.

Methods

Every inward UTI episode, culture and antimicrobial susceptibility was done while on past 6-month, history of infections and use of antimicrobials was collected using clinical records and demonstration of antimicrobials. Children with recurrent pyelonephritis was followed and in vitro bio film formation was assessed.

Results

Frequency of UTI was significantly high among infants (p?=?0.03). Last 6-month, all (220) were exposed to antimicrobials. Cefixime was the commonly prescribed antimicrobial (p?=?0.02). In current UTI episode, 64.5% (142/220) of children with UTI were consulted GPs’ prior to seek treatment from tertiary care pediatric unit (p?=?0.02). While on follow up child who developed UTI, found urine culture isolates were significantly shifted from E. coli and K. pneumoniae to extended spectrum of beta-lactamase (ESBL) E. coli and K. pneumoniae. Out of 208 participants, 36 of them had re-current pyelonephritis (R-PN). Renal scarring (RS) was detected in 22 out of 70 patients with pyelonephritis following dimercaptosuccinic acid scan. Following each episodes of recurrent pyelonephritis 11% of new scar formation was detected (p?=?0.02). Bio film forming E. coli and K. pneumoniae was significantly associated in patients with R-PN (p?=?0.04).

Discussion

Medical care providers often prescribe antimicrobials without having an etiological diagnosis. While continuing exposure of third generation cephalosporin and carbapenem leads to development of ESBL and CRE microbes in great. The empiric uses of antimicrobials need to be stream lined with local epidemiology and antimicrobial susceptibility pattern. R-PN in childhood leads to RS. In great, bio film formation act as the focus for such recurrences.

     

Incidence, risk factors and mortality of nosocomial pneumonia in Intensive Care Units: A prospective study

Background

Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.

Methods

In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).

Results and discussion

In total, 58.0%, 66.1%, and 55.8% of S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates (128 isolates were resistant to penicillin [MIC >= 2 mg/L], 72 intermediate [MIC 0.12–1 mg/L]) while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis (MIC₅₀ = 0.06 mg/L, MIC₉₀ = 0.12 mg/L).

Conclusion

The high in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.     

Prevalence and Antimicrobial Resistance of Microbes Causing Bloodstream Infections in Unguja,Zanzibar

Background

Bloodstream infections (BSI) are frequent and cause high case-fatality rates. Urgent antibiotic treatment can save patients’ lives, but antibiotic resistance can render antibiotic therapy futile. This study is the first to collect epidemiological data on BSI from Unguja, Zanzibar.

Methods

Clinical data and blood for culturing and susceptibility testing of isolated microbes were obtained from 469 consecutively enrolled neonates, children and adults presenting with signs of systemic infections at Mnazi Mmoja Hospital (MMH), Zanzibar.

Results

Pathogenic bacteria were recovered from the blood of 14% of the patients (66/469). The most frequently isolated microbes were Klebsiella pneumoniae, Escherichia coli, Acinetobacter spp. and Staphylococcus aureus. Infections were community-acquired in 56 patients (85%) and hospital-acquired in 8 (12%) (data missing for 2 patients). BSI caused by extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae (E. coli, K. pneumoniae) was found in 5 cases, of which 3 were community-acquired and 2 hospital-acquired. Three of these patients died. Six of 7 Salmonella Typhi isolates were multidrug resistant. Streptococcus pneumoniae was found in one patient only.

Conclusions

This is the first report of ESBL-producing bacteria causing BSI from the Zanzibar archipelago. Our finding of community-acquired BSI caused by ESBL-producing bacteria is alarming, as it implies that these difficult-to-treat bacteria have already spread in the society. In the local setting these infections are virtually impossible to cure. The findings call for increased awareness of rational antibiotic use, infection control and surveillance to counteract the problem of emerging antimicrobial resistance.     

Empiric Antibiotic Therapy for Staphylococcus aureus Bacteremia May Not Reduce In-Hospital Mortality: A Retrospective Cohort Study

Background

Appropriate empiric therapy, antibiotic therapy with in vitro activity to the infecting organism given prior to confirmed culture results, may improve Staphylococcus aureus outcomes. We aimed to measure the clinical impact of appropriate empiric antibiotic therapy on mortality, while statistically adjusting for comorbidities, severity of illness and presence of virulence factors in the infecting strain.

Methodology

We conducted a retrospective cohort study of adult patients admitted to a tertiary-care facility from January 1, 2003 to June 30, 2007, who had S. aureus bacteremia. Time to appropriate therapy was measured from blood culture collection to the receipt of antibiotics with in vitro activity to the infecting organism. Cox proportional hazard models were used to measure the association between receipt of appropriate empiric therapy and in-hospital mortality, statistically adjusting for patient and pathogen characteristics.

Principal Findings

Among 814 admissions, 537 (66%) received appropriate empiric therapy. Those who received appropriate empiric therapy had a higher hazard of 30-day in-hospital mortality (Hazard Ratio (HR): 1.52; 95% confidence interval (CI): 0.99, 2.34). A longer time to appropriate therapy was protective against mortality (HR: 0.79; 95% CI: 0.60, 1.03) except among the healthiest quartile of patients (HR: 1.44; 95% CI: 0.66, 3.15).

Conclusions/Significance

Appropriate empiric therapy was not associated with decreased mortality in patients with S. aureus bacteremia except in the least ill patients. Initial broad antibiotic selection may not be widely beneficial.     

Effect of age and vaccination on extent and spread of Chlamydia pzneumoniae infection in C57BL/6 mice

Background

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen for humans. Infection by C. pneumoniae may be linked etiologically to extra-respiratory diseases of aging, especially atherosclerosis. We have previously shown that age promotes C. pneumoniae respiratory infection and extra-respiratory spread in BALB/c mice.

Findings

Aged C57BL/6 mice had a greater propensity to develop chronic and/or progressive respiratory infections following experimental intranasal infection by Chlamydia pneumoniae when compared to young counterparts. A heptavalent CTL epitope minigene (CpnCTL7) vaccine conferred equal protection in the lungs of both aged and young mice. This vaccine was partially effective in protecting against C. pneumoniae spread to the cardiovascular system of young mice, but failed to provide cardiovascular protection in aged animals.

Conclusions

Our findings suggest that vaccine strategies that target the generation of a C. pneumoniae-specific CTL response can protect the respiratory system of both young and aged animals, but may not be adequate to prevent dissemination of C. pneumoniae to the cardiovascular system or control replication in those tissues in aged animals.     

A set of novel multiplex Taqman real-time PCRs for the detection of diarrhoeagenic Escherichia coli and its use in determining the prevalence of EPEC and EAEC in a university hospital

Background

Escherichia coli isolates of equine faecal origin were investigated for antibiotic resistance, resistance genes and their ability to perform horizontal transfer.

Methods

In total, 264 faecal samples were collected from 138 horses in hospital and community livery premises in northwest England, yielding 296 resistant E. coli isolates. Isolates were tested for susceptibility to antimicrobial drugs by disc diffusion and agar dilution methods in order to determine minimum inhibitory concentrations (MIC). PCR amplification was used to detect genes conferring resistance to: ampicillin (TEM and SHV beta-lactamase), chloramphenicol (catI, catII, catIII and cml), tetracycline (tetA, tetB, tetC, tetD, tet E and tetG), and trimethoprim (dfrA1, dfrA9, dfrA12, dfrA13, dfr7, and dfr17).

Results

The proportion of antibiotic resistant isolates, and multidrug resistant isolates (MDR) was significantly higher in hospital samples compared to livery samples (MDR: 48% of hospital isolates; 12% of livery isolates, p < 0.001). Resistance to ciprofloxacin and florfenicol were identified mostly within the MDR phenotypes. Resistance genes included dfr, TEM beta-lactamase, tet and cat, conferring resistance to trimethoprim, ampicillin, tetracycline and chloramphenicol, respectively. Within each antimicrobial resistance group, these genes occurred at frequencies of 93% (260/279), 91%, 86.8% and 73.5%, respectively; with 115/296 (38.8%) found to be MDR isolates. Conjugation experiments were performed on selected isolates and MDR phenotypes were readily transferred.

Conclusions

Our findings demonstrate that E. coli of equine faecal origin are commonly resistant to antibiotics used in human and veterinary medicine. Furthermore, our results suggest that most antibiotic resistance observed in equine E. coli is encoded by well-known and well-characterized resistant genes common to E. coli from man and domestic animals. These data support the ongoing concern about antimicrobial resistance, MDR, antimicrobial use in veterinary medicine and the zoonotic risk that horses could potentially pose to public health.     

Sequence Analysis of pKF3-70 in Klebsiella pneumoniae: Probable Origin from R100-Like Plasmid of Escherichia coli

Background

Klebsiella pneumoniae is a clinically significant species of bacterium which causes a variety of diseases. Clinical treatment of this bacterial infection is greatly hindered by the emergence of multidrug-resistant strains. The resistance is largely due to the acquisition of plasmids carrying drug-resistant as well as pathogenic genes, and its conjugal transfer facilitates the spread of resistant phenotypes.

Methodology/Principal Findings

The 70,057 bp plasmid pKF3-70, commonly found in Klebsiella pneumoniae, is composed of five main functional modules, including regions involved in replication, partition, conjugation, transfer leading, and variable regions. This plasmid is more similar to several Escherichia coli plasmids than any previously reported K. pneumoniae plasmids and pKF3-70 like plasmids share a common and conserved backbone sequence. The replication system of the pKF3-70 is 100% identical to that of RepFII plasmid R100 from E. coli. A beta-lactamase gene ctx-m-14 with its surrounding insertion elements (ISEcp1, truncated IS903 and a 20 bp inverted repeat sequence) may compose an active transposon which is directly bordered by two putative target repeats “ATTAC.”

Conclusions/Significance

The K. pneumoniae plasmid pKF3-70 carries an extended-spectrum beta-lactamase gene, ctx-m-14. The conjugative characteristic makes it a widespread plasmid among genetically relevant genera which poses significant threat to public health.     

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

Background

Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.

Methods

Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.

Results

In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI ?92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI ?57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day.

Conclusions

In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.     

Emerging resistance among bacterial pathogens in the intensive care unit – a European and North American Surveillance study (2000–2002)

Background

Globally ICUs are encountering emergence and spread of antibiotic-resistant pathogens and for some pathogens there are few therapeutic options available.

Methods

Antibiotic in vitro susceptibility data of predominant ICU pathogens during 2000–2 were analyzed using data from The Surveillance Network (TSN) Databases in Europe (France, Germany and Italy), Canada, and the United States (US).

Results

Oxacillin resistance rates among Staphylococcus aureus isolates ranged from 19.7% to 59.4%. Penicillin resistance rates among Streptococcus pneumoniae varied from 2.0% in Germany to as high as 20.2% in the US; however, ceftriaxone resistance rates were comparably lower, ranging from 0% in Germany to 3.4% in Italy. Vancomycin resistance rates among Enterococcus faecalis were ≤ 4.5%; however, among Enterococcus faecium vancomycin resistance rates were more frequent ranging from 0.8% in France to 76.3% in the United States. Putative rates of extended-spectrum β-lactamase (ESBL) production among Enterobacteriaceae were low, <6% among Escherichia coli in the five countries studied. Ceftriaxone resistance rates were generally lower than or similar to piperacillin-tazobactam for most of the Enterobacteriaceae species examined. Fluoroquinolone resistance rates were generally higher for E. coli (6.5% – 13.9%), Proteus mirabilis (0–34.7%), and Morganella morganii (1.6–20.7%) than other Enterobacteriaceae spp (1.5–21.3%). P. aeruginosa demonstrated marked variation in β-lactam resistance rates among countries. Imipenem was the most active compound tested against Acinetobacter spp., based on resistance rates.

Conclusion

There was a wide distribution in resistance patterns among the five countries. Compared with other countries, Italy showed the highest resistance rates to all the organisms with the exception of Enterococcus spp., which were highest in the US. This data highlights the differences in resistance encountered in intensive care units in Europe and North America and the need to determine current local resistance patterns by which to guide empiric antimicrobial therapy for intensive care infections.     

Mycobacterium tuberculosis Bacteremia in a Cohort of HIV-Infected Patients Hospitalized with Severe Sepsis in Uganda–High Frequency,Low Clinical Sand Derivation of a Clinical Prediction Score

Background

When manifested as Mycobacterium tuberculosis (MTB) bacteremia, disseminated MTB infection clinically mimics other serious blood stream infections often hindering early diagnosis and initiation of potentially life-saving anti-tuberculosis therapy. In a cohort of hospitalized HIV-infected Ugandan patients with severe sepsis, we report the frequency, management and outcomes of patients with MTB bacteremia and propose a risk score based on clinical predictors of MTB bacteremia.

Methods

We prospectively enrolled adult patients with severe sepsis at two Ugandan hospitals and obtained blood cultures for MTB identification. Multivariable logistic regression modeling was used to determine predictors of MTB bacteremia and to inform the stratification of patients into MTB bacteremia risk categories based on relevant patient characteristics.

Results

Among 368 HIV-infected patients with a syndrome of severe sepsis, eighty-six (23%) had MTB bacteremia. Patients with MTB bacteremia had a significantly lower median CD4 count (17 vs 64 lymphocytes/mm³, p<0.001) and a higher 30-day mortality (53% vs 32%, p = 0.001) than patients without MTB bacteremia. A minority of patients with MTB bacteremia underwent standard MTB diagnostic testing (24%) or received empiric anti-tuberculosis therapy (15%). Independent factors associated with MTB bacteremia included male sex, increased heart rate, low CD4 count, absence of highly active anti-retroviral therapy, chief complaint of fever, low serum sodium and low hemoglobin. A risk score derived from a model containing these independent predictors had good predictive accuracy [area under the curve = 0.85, 95% CI 0.80–0.89].

Conclusions

Nearly 1 in 4 adult HIV-infected patients hospitalized with severe sepsis in 2 Ugandan hospitals had MTB bacteremia. Among patients in whom MTB was suspected, standard tests for diagnosing pulmonary MTB were inaccurate for correctly classifying patients with or without bloodstream MTB infection. A MTB bacteremia risk score can improve early diagnosis of MTB bacteremia particularly in settings with increased HIV and MTB co-infection.     

Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait

Background

Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples.

Objective

The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay.

Methods

A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea).

Results

Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool.

Conclusion

The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.     

Genotypic and metabolic approaches towards the segregation of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strains producing different antibiotic resistant enzymes

Introduction

Genotype and metabolomic variation are important for bacterial survival and adaptation to environmental changes.

Objectives

In this study, we compared the relationship among Klebsiella pneumoniae strains based on their genotypic and metabolic profiles. In addition, we also evaluated the association of the relationship with beta-lactamase production.

Methods

A total of 53 K. pneumoniae strains isolated in 2013–2014 from a tertiary teaching hospital in Malaysia were subjected to antimicrobial susceptibility testing (AST) via disk diffusion method and beta-lactamase production confirmation. The bacterial strains were also typed genotypically and metabolically via REP-PCR and ¹H-NMR spectroscopy respectively. The concordance of the matrices derived based on genotypic and metabolic characterization was measured based on Spearman’s rank correlation.

Results

Spearman’s correlation rank showed that there is a weak but significant negative correlation between the genetic fingerprints and metabolic profiles of K. pneumoniae. Specifically, K. pneumoniae strains were clustered into five major clusters based on REP-PCR where most of the carbapenem resistant K. pneumoniae (CRKP) strains made up the major cluster. In contrast, metabolic patterns of the three groups (i.e. CRKP, extended spectrum beta-lactamase producing K. pneumoniae (ESBL), susceptible) of K. pneumoniae were clearly differentiated on PLS-DA score plots derived from ¹H-NMR spectroscopy.

Conclusion

Overall, this study showed that metabolomic profiling using ¹H-NMR spectroscopy is able to discriminate K. pneumoniae strains based on their beta-lactamase production status.

     

A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples

Background

Streptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections.

Results

We have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 2^8-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 μg/mL kanamycin.

Conclusions

This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity.     

Bacteremia in Children Hospitalized with Respiratory Syncytial Virus Infection

Background

The risk of bacteremia is considered low in children with acute bronchiolitis. However the rate of occult bacteremia in infants with RSV infection is not well established. The aim was to determine the actual rate and predictive factors of bacteremia in children admitted to hospital due to confirmed RSV acute respiratory illness (ARI), using both conventional culture and molecular techniques.

Methods

A prospective multicenter study (GENDRES-network) was conducted between 2011–2013 in children under the age of two admitted to hospital because of an ARI. Among those RSV-positive, bacterial presence in blood was assessed using PCR for Meningococcus, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, in addition to conventional cultures.

Results

66 children with positive RSV respiratory illness were included. In 10.6% patients, bacterial presence was detected: H. influenzae (n = 4) and S. pneumoniae (n = 2). In those patients with bacteremia, there was a previous suspicion of bacterial superinfection and had received empirical antibiotic treatment 6 out of 7 (85.7%) patients. There were significant differences in terms of severity between children with positive bacterial PCR and those with negative results: PICU admission (100% vs. 50%, P-value = 0.015); respiratory support necessity (100% vs. 18.6%, P-value < 0.001); Wood-Downes score (mean = 8.7 vs. 4.8 points, P-value < 0.001); GENVIP scale (mean = 17 vs. 10.1, P-value < 0.001); and length of hospitalization (mean = 12.1 vs. 7.5 days, P-value = 0.007).

Conclusion

Bacteremia is not frequent in infants hospitalized with RSV respiratory infection, however, it should be considered in the most severe cases.     

Detection of Streptococcus pneumoniae from Different Types of Nasopharyngeal Swabs in Children

Background

A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs.

Methods

The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA-targeted real-time polymerase chain reaction (qPCR).

Results

Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5–16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10⁴ CFU/mL [IQR, 2.0×10² – 4.0×10⁵ CFU/mL]) than Dacron swabs (3.7×10⁴ CFU/mL [IQR, 4.0×10²–3.2×10⁵ CFU/mL], p = 0.17). Using lytA-targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10⁵ genome copies/mL [IQR, 1.3×10²−1.8×10⁶] vs. 9.3×10⁴ genome copies/mL [IQR, 7.0×10¹−1.1×10⁶]; p = 0.005).

Conclusion

Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.     

Persistent Catheter-Related Staphylococcus aureus Bacteremia after Catheter Removal and Initiation of Antimicrobial Therapy

Objectives

Catheter-related Staphylococcus aureus bacteremia (CRSAB) occasionally persists despite catheter removal and initiation of appropriate antimicrobial therapy. The aim of this study was to determine the incidence, risk factors, and outcomes of persistent CRSAB after catheter removal and initiation of antimicrobial therapy.

Methods

Consecutive patients with CRSAB were prospectively included from over a 41-month period. We compared the clinical features, 40 bacterial virulence genes, and outcomes between patients with persistent CRSAB (i.e., bacteremia for >3 days after catheter removal and initiation of appropriate antimicrobial therapy) and non-persistent CRSAB.

Results

Among the 220 episodes of CRSAB, the catheter was kept in place in 17 (6%) and removed in 203 (94%) cases. In 43 (21%) of the 203 episodes, bacteremia persisted for >3 days after catheter removal and initiation of antimicrobial therapy. Methicillin resistance (Odds ratio [OR], 9.01; 95% confidence interval [CI], 3.05–26.61; P<0.001), non-catheter prosthetic devices (OR, 5.37; 95% CI, 1.62–17.80; P = 0.006), and renal failure (OR, 3.23; 95% CI, 1.48–7.08; P = 0.003) were independently associated with persistent CRSAB. Patients with persistent CRSAB were more like to experience complication than were those with non-persistent CRSAB (72% vs. 15%; P<0.001). Among all episodes due to methicillin-resistant S. aureus, persistent CRSAB isolates were associated with accessory gene regulator (agr) group II (P = .04), but presence of other bacterial virulence genes, distribution of vancomycin minimum inhibitory concentration distribution, and frequency of vancomycin heteroresistance did not differ between the groups.

Conclusions

In patients with CRSAB, bacteremia persisted in 21% of cases despite catheter removal and initiation of antimicrobial therapy. Methicillin resistance, renal failure, and non-catheter prosthetic devices were independent risk factors for persistent CRSAB, which was associated with a higher rate of complications.     

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae

Background

Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.

Results

The alanine racemases gene from S. pneumoniae (alr _SP ) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg^-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.

Conclusion

We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.     

Biofilm formation and adherence characteristics of an Elizabethkingia meningoseptica isolate from Oreochromis mossambicus

Background

The aim of this study was to describe a novel trimethoprim resistance gene cassette, designated dfrA30, within a class 1 integron in a facultatively oligotrophic, multiple antibiotic and human serum resistant test strain, MB45, in a population of oligotrophic bacteria isolated from the river Mahananda; and to test the efficiency of surface bound acetate on zinc oxide quantum dots (ZnO QDs) as bactericidal agent on MB45.

Methods

Diluted Luria broth/Agar (10^-3) media was used to cultivate the oligotrophic bacteria from water sample. Multiple antibiotic resistant bacteria were selected by employing replica plate method. A rapid assay was performed to determine the sensitivity/resistance of the test strain to human serum. Variable region of class 1 integron was cloned, sequenced and the expression of gene coding for antibiotic resistance was done in Escherichia coli JM 109. Identity of culture was determined by biochemical phenotyping and 16S rRNA gene sequence analyses. A phylogenetic tree was constructed based on representative trimethoprim resistance-mediating DfrA proteins retrieved from GenBank. Growth kinetic studies for the strain MB45 were performed in presence of varied concentrations of ZnO QDs.

Results and conclusions

The facultatively oligotrophic strain, MB45, resistant to human serum and ten antibiotics trimethoprim, cotrimoxazole, ampicillin, gentamycin, netilmicin, tobramycin, chloramphenicol, cefotaxime, kanamycin and streptomycin, has been identified as a new strain of Klebsiella pneumoniae. A novel dfr gene, designated as dfrA30, found integrated in class 1 integron was responsible for resistance to trimethoprim in Klebsiella pneumoniae strain MB45. The growth of wild strain MB45 was 100% arrested at 500 mg/L concentration of ZnO QDs. To our knowledge this is the first report on application of ZnO quantum dots to kill multiple antibiotics and serum resistant K. pneumoniae strain.     

Severe Sepsis in Two Ugandan Hospitals: a Prospective Observational Study of Management and Outcomes in a Predominantly HIV-1 Infected Population

Background

Sepsis likely contributes to the high burden of infectious disease morbidity and mortality in low income countries. Data regarding sepsis management in sub-Saharan Africa are limited. We conducted a prospective observational study reporting the management and outcomes of severely septic patients in two Ugandan hospitals. We describe their epidemiology, management, and clinical correlates for mortality.

Methodology/Results

Three-hundred eighty-two patients fulfilled enrollment criteria for a severe sepsis syndrome. Vital signs, management and laboratory results were recorded. Outcomes measured included in-hospital and post-discharge mortality.Most patients were HIV-infected (320/377, 84.9%) with a median CD4+ T cell (CD4) count of 52 cells/mm³ (IQR, 16–131 cells/mm³). Overall mortality was 43.0%, with 23.7% in-hospital mortality (90/380) and 22.3% post-discharge mortality (55/247). Significant predictors of in-hospital mortality included admission Glasgow Coma Scale and Karnofsky Performance Scale (KPS), tachypnea, leukocytosis and thrombocytopenia. Discharge KPS and early fluid resuscitation were significant predictors of post-discharge mortality. Among HIV-infected patients, CD4 count was a significant predictor of post-discharge mortality.Median volume of fluid resuscitation within the first 6 hours of presentation was 500 mLs (IQR 250–1000 mls). Fifty-two different empiric antibacterial regimens were used during the study. Bacteremic patients were more likely to die in hospital than non-bacteremic patients (OR 1.83, 95% CI = 1.01–3.33). Patients with Mycobacterium tuberculosis (MTB) bacteremia (25/249) had higher in-hospital mortality (OR 1.97, 95% CI = 1.19–327) and lower median CD4 counts (p = 0.001) than patients without MTB bacteremia.

Conclusion

Patients presenting with sepsis syndromes to two Ugandan hospitals had late stage HIV infection and high mortality. Bacteremia, especially from MTB, was associated with increased in-hospital mortality. Most clinical predictors of in-hospital mortality were easily measurable and can be used for triaging patients in resource-constrained settings. Procurement of low cost and high impact treatments like intravenous fluids and empiric antibiotics may help decrease sepsis-associated mortality in resource-constrained settings.