Commensal Escherichia coli isolates from healthy zoo animals kept in Ostrava Zoological Garden, Czech Republic, were investigated to evaluate the dissemination of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. A total of 160 faecal samples of various animal species were inoculated onto MacConkey agar with cefotaxime (2 mg L(-1) ) or ciprofloxacin (0.05 mg L(-1) ) to obtain ESBL- or PMQR-positive E. coli isolates. Clonality of E. coli isolates was investigated by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Plasmids carrying ESBL or PMQR genes were typed by PCR-based replicon typing, plasmid MLST and restriction fragment length polymorphism. Forty-nine (71%, n = 69) cefotaxime-resistant and 15 (16%, n = 94) ciprofloxacin-resistant E. coli isolates harboured ESBL or PMQR genes. Isolates were assigned to 18 sequence types (ST) and 20 clusters according to their macrorestriction patterns by PFGE. The genes blaCTX-M-1 and qnrS1 were detected on highly related IncI1 plasmids assigned to clonal complex 3 (ST3, ST38) and on non-related IncN plasmids of ST1 and ST3, respectively. The gene qnrS1 was located on related IncX1 plasmids. Dissemination of antibiotic resistance is associated with spreading of particular E. coli clones and plasmids of specific incompatibility groups among various animal species. This article is protected by copyright. All rights reserved.

Understanding epistasis is central to biology. For instance, epistatic interactions determine the topography of the fitness landscape and affect the dynamics and determinism of adaptation. However, few empirical data are available and comparing results is complicated by confounding variation in the system and the type of mutations used. Here, we take a systematic approach by quantifying epistasis in two sets of four beneficial mutations in the antibiotic resistance enzyme TEM-1 β-lactamase. Mutations in these sets either have large or small effects on cefotaxime resistance when present as single mutations. By quantifying the epistasis and ruggedness in both landscapes we find two general patterns. First, resistance is maximal for combinations of two mutations in both fitness landscapes and declines when more mutations are added due to abundant sign epistasis and a pattern of diminishing returns with genotype resistance. Second, large-effect mutations interact more strongly than small-effect mutations, suggesting that the effect size of mutations may be an organizing principle in understanding patterns of epistasis. By fitting the data to simple phenotype-resistance models, we show that this pattern may be explained by the nonlinear dependence of resistance on enzyme stability and an unknown phenotype when mutations have antagonistically pleiotropic effects. The comparison to a previously published set of mutations in the same gene with a joint benefit further shows that the enzyme's fitness landscape is locally rugged, but does contain adaptive pathways that lead to high resistance.

OBJECTIVES:

The aim of this study was to evaluate the accuracies of these automated susceptibility test systems with cefepime, cefotaxime and ceftazidime using the new CLSI and EUCAST guidelines in extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae.

METHODS:

A total of 220 ESBL-producing clinical isolates were collected from 12 hospitals in Korea. Susceptibility testing for cefepime, cefotaxime and ceftazidime was performed by MicroScan WalkAway, Vitek 2 and the CLSI broth microdilution test. ESBL genotypes were determined by PCR amplification.

RESULTS:

The proportion of isolates classified as susceptible to cefepime and ceftazidime with the CLSI and EUCAST guidelines was 35.0% versus 2.3% for cefepime (P < 0.001) and 21.8% versus 8.2% for ceftazidime (P < 0.001), respectively, and the susceptible isolates were mainly the CTX-M-9 group or SHV-type ESBL producers. All of the isolates were resistant to cefotaxime. Against the total of 220 ESBL-producing isolates, using the CLSI (EUCAST) criteria, very major/major error rates of MicroScan and Vitek 2 were as follows: 1.9%/20.8% (1.8%/20.0%) and 27.4%/0% (12.2%/0%) for cefepime and 2.6%/8.3% (1.2%/0%) and 4.5%/0% (2.3%/0%) for ceftazidime, respectively. The very major error rates of MicroScan and Vitek 2 with cefotaxime were 0.9% and 1.4%, respectively. The errors were mainly major errors for MicroScan and very major errors for Vitek 2.

CONCLUSIONS:

A substantial portion of ESBL-producing isolates were susceptible to cefepime and ceftazidime by using the CLSI and EUCAST breakpoints. Unfortunately, the error rates of the two automated susceptibility systems were not acceptable for cefepime and ceftazidime.

OPINION STATEMENT: The tick-borne spirochete responsible for Lyme disease is highly antibiotic-sensitive. Treatment related misconceptions can be attributed to confusion in three principal realms: (1) the appropriate approach to diagnosis (who should be treated); (2) necessary and appropriate treatment; and (3) what actually constitutes nervous system infection and to what extent this mandates different treatment. Contrary to often-repeated assertions, laboratory-based diagnosis-in the appropriate setting-is as valid as it is in most other serologically diagnosed infections. Treatment is highly effective in the vast majority of patients, including those with nervous system disease. Nervous system infection, most typically meningitis, cranial neuritis, radiculoneuritis, and other forms of mononeuropathy multiplex, is highly antibiotic responsive. The encephalopathy that can be seen in some patients with active infection represents the same phenomenon that occurs in patients with many other inflammatory disorders, is not evidence of central nervous system (CNS) infection, and does not require any different, more prolonged, or more intensive treatment. In patients with infection not involving the CNS, oral treatment with amoxicillin, cefuroxime axetil, or doxycycline for 2-4 weeks is almost always curative. Despite historic preferences for parenteral treatment with ceftriaxone, cefotaxime, or meningeal dose penicillin, patients with the forms of nervous system involvement listed above are highly responsive to oral doxycycline. Parenteral regimens can be reserved for those very rare patients with parenchymal CNS involvement, other severe forms of infection, or the approximately 5 % of patients who fail to respond to oral regimens.

To determine the common aetiolog of acute bacterial meningitis in children and their antibiotic susceptibility pattern.A retrospective study with a review of cerebrospinal fluid culture reports of paediatric patients aged 0-15 years, suspected of acute meningitis in the Medical Microbiology Department of Aminu Kano Teaching Hospital, Kano, Nigeria from October 2006 to October 2009 from October 2006 to October 2009.A positive culture bacterial isolation rate of 3.3% (n=50/1500) with prevalence of Streptococcus pneumoniae (24%), Neisseria meningitidis(22%), Escherichia coli(16%), Haemophilus influenzae (14%), Group B streptococci(8%) and Enterococci(8%) which were susceptible to ceftriaxone(96%), cefotaxime(95%) and ciprofloxacin(93%) across the bacterial isolates. Neonates were 55% (n=6.8/12.4) most at risk.Neonates are the most at risk of acute bacterial meningitis. In the absence of antibiotic susceptibility report, ceftriaxone should be considered as a first choice reliable antibiotic for empirical treatment of meningitis in children, in this environment.

Burkholderia cepacia complex (Bcc) and Burkholderia pseudomallei (Bpm) are opportunistic-human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g., PenA in Bcc and PenI in Bpm). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor- resistant carbapenemase, most similar to KPC-2 (the most clinically-significant serine carbapenemase) while PenI is an extended-spectrum β-lactamase, ESBL. PenA hydrolyzes β-lactams with kcat values ranging from 0.38 +/- 0.04 to 460 +/- 46 s-1 and possesses high kcat/kinact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI, the ESBL, demonstrates the highest kcat value for cefotaxime of 9.0 +/- 0.9 s-1. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explains the lower kcat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. On the other hand, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.

OBJECTIVES:

Several studies on faecal carriage of extended-spectrum β-lactamase (ESBL)/AmpC-producing Escherichia coli have been performed in cattle, but little is known about faecal carriage in veal calves. This study describes the prevalence and molecular characteristics of ESBL/AmpC genes in E. coli isolated from faecal samples of veal calves from 1997 to 2010.

METHODS:

Pooled faecal samples were inoculated using selective enrichment broth and subsequently selective MacConkey agar. All isolates with reduced susceptibility to cefotaxime were screened by PCR and sequencing analysis for the presence of ESBL/AmpC genes.

RESULTS:

The prevalence of E. coli with reduced susceptibility to cefotaxime showed a discontinuous increasing trend, ranging from 4% in 1998 and 1999 to 39% in 2010. Promoter mutations of the chromosomal ampC gene were present in all years. In 2000, ESBL genes blaCTX-M-1, blaTEM-52 and blaTEM-20 were first observed. Before 2005 the majority of E. coli with reduced susceptibility to cefotaxime harboured ampC promoter mutations. From 2005 onwards the majority harboured blaCTX-M genes, of which blaCTX-M-1 was the most abundant, followed by blaCTX-M-14 and blaCTX-M-15. The diversity of blaCTX-M genes gradually increased from one variant in 2000 to six variants in 2010. The prevalence of blaTEM-52 was relatively low, but it was detected from 2000 onwards. blaCMY and blaSHV were found sporadically.

CONCLUSIONS:

The prevalence and molecular diversity of genes encoding cefotaxime resistance in E. coli isolated from veal calves over a 14 year period showed an increasing trend. From 2005 onwards, blaCTX-M genes were most abundant, especially blaCTX-M-1.

We investigated changes in serotypes and antimicrobial susceptibilities among 386 isolates of invasive Streptococcus pneumoniae collected from numerous hospitals in Korea from 1996-2008. Serotype 19F (9.8%), 23F (8.3%), 19A (7.8%), 6A (7.5%), 3 (7.3%), 9V (6.5%), 6B (6.2%), 14 (4.9%), 1 (3.9%), 11A (3.9%) and 4 (3.1%) represented 69.2% of all isolates. While the overall proportion of PCV7 serotypes was stable over time, we observed modest decreases in children < 5-years-of-age and adults ≥ 65-years-of-age between 1996-1999 and 2007-2008. An increased prevalence of non-PCV7 serotypes in these age groups was primarily attributable to an increase in serotypes 3, 6A and 19A. Most invasive S. pneumoniae isolates showed high resistance rates to erythromycin (74.9%), tetracycline (71.1%) and clindamycin (61.7%). From 1996-2003 and 2004-2008, non-susceptibility rates to cefotaxime and multi-drugs (three or more classes) in PCV7 serotypes showed a declining trend, while in non-PCV7 serotypes there was an increasing trend. Non-PCV7 serotypes 6A and 19A, which mostly exhibited multidrug-resistant phenotypes (69.0% and 76.7% respectively), increased between 1996-2003 and 2004-2008. Although PCV7 was introduced in Korea in November 2003, pneumococcal vaccination has not been included in the national child vaccination program. Our results provide serotype coverage useful to consider the adoption of universal pneumococcal vaccination in Korea.

BACKGROUND:

Antibiotic resistance is one of the most serious public health concerns worldwide and is increasing at an alarming rate, making daily treatment decisions more challenging. This study is aimed at identifying local bacterial isolates and their antimicrobial susceptibility patterns to avoid irrational antibiotic use, especially in settings where unguided management occurs and febrile illnesses are predominant.

MATERIAL AND METHODS:

A hospital-based prospective cross-sectional study was conducted from September 2011 to February 2012. Febrile children were serially recruited and demographic and clinical data were collected using a standardized data collection tool. A blood culture was performed and identification of the isolates was undertaken using in-house biochemical tests. Susceptibility to common antibiotics was investigated using the disc diffusion methods.

RESULTS:

Of the 1081 children admitted during the study period, 317 (29.3%) met the inclusion criteria and were recruited, of whom 195 (61.5%) and 122 (38.5%) were male and female respectively. The median age was 18 months with an interquartile range of 9 to 36 months. Of the 317 children, 251 (79.2%) were below or equal to 36 months of age. The prevalence of bacteremia was 6.6%. A higher prevalence of bacteraemia was observed in children below 36 months than in those >= 36 months (7.5% vs. 3.0%, p = 0.001). Predictors of bacteraemia were an axillary temperature of >38.5 [degree sign]C (OR =7, 95% CI = 2.2 - 14.8, p-value = 0.0001), a positive malaria slide (OR =5, 95% CI = 3.0 - 21.2, p-value = 0.0001) and a high neutrophils' count (OR =21 95% CI = 5.6 - 84, p-value = 0.0001). Escherichia coli and Klebsiella pneumoniae accounted for 7 (33.3%) and 6 (28.6%) of all the isolates respectively. Others gram-negatives bacteria were Citrobacter spp 2 (9.5%), Enterobacter spp 1 (4.25%), Pseudomonas spp 2 (9.5%), Proteus spp 1 (4.25%) and Salmonella spp 1 (4.25%). These isolates were highly resistant to ampicillin (95%), co-trimoxazole (90%), tetracycline (90%), gentamicin (80%), augmentin (80%), chloramphenicol (65%), ceftriaxone (35%), cefotaxime (35%) ciprofloxacin (30%), amikacin (30%), ceftazidime (25%) and norfloxacine (10%).

CONCLUSION:

Multi-resistant gram-negative bacteria are the commonest cause of bacteremia in under-fives attending the Bugando Medical Centre, Mwanza, Tanzania. A high body temperature, a positive malaria slide and a high absolute neutrophils' count were all independent risk factors found to predict bacteremia. A higher mortality rate was observed in children with bacteraemia. Continuous epidemiological surveillance should be conducted so that a proper and effective antibiotics management can be instituted, especially in children with a high grade fever, a positive malaria slide and a high neutrophils' count.

Organic anion transporter 3 (OAT3, SLC22A8), a transporter expressed on the basolateral membrane of the proximal tubule, plays a critical role in the renal excretion of organic anions including many therapeutic drugs. The goal of this study was to evaluate the in vivo effects of the OAT3-Ile305Phe variant (rs11568482), present at 3.5% allele frequency in Asians, on drug disposition with a focus on cefotaxime, a cephalosporin antibiotic. In HEK293-Flp-In cells, the OAT3-Ile305Phe variant had a lower maximum cefotaxime transport activity, Vmax , [159 ± 3 nmol*(mg protein)(-1) /min (mean ± SD)] compared with the reference OAT3 [305 ± 28 nmol*(mg protein)(-1) /min, (mean ± SD), p < 0.01], whereas the Michaelis-Menten constant values (Km ) did not differ. In healthy volunteers, we found volunteers that were heterozygous for the Ile305Phe variant and had a significantly lower cefotaxime renal clearance (CLR ; mean ± SD: 84.8 ± 32.1 mL/min, n = 5) compared with volunteers that were homozygous for the reference allele (158 ± 44.1 mL/min, n = 10; p = 0.006). Furthermore, the net secretory component of cefotaxime renal clearance (CLsec ) was reduced in volunteers heterozygous for the variant allele [33.3 ± 31.8 mL/min (mean ± SD)] compared with volunteers homozygous for the OAT3 reference allele [97.0 ± 42.2 mL/min (mean ± SD), p = 0.01]. In summary, our study suggests that a low-frequency reduced-function polymorphism of OAT3 associates with reduced cefotaxime CLR and CLsec . © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.