Ceftazidime Resistance Research Articles

Detection of AmpC β-lactamase producing bacteria isolated in neonatal sepsis.

Objective:The objective of this study was to determine the occurrence and antimicrobial profile of AmpC β-lactamase producing bacteria.Methods:The study was conducted at The Children’s Hospital and The Institute of Child Health Lahore, Pakistan, during September 2011 to June 2012. A total number of 1,914 blood samples of suspected neonatal septicemia were processed. Isolates were identified using Gram’s staining, API 20E and API 20NE tests. Gram negative isolates were screened for AmpC β-lactamase production against ceftazidime, cefotaxime and cefoxitin resistance and confirmed by inhibitor based method.Results:Total number of 54 (8.49%) Gram positive and 582 (91.5%) Gram negative bacteria were identified. Among Gram negative isolates 141 (22%) were AmpC producers and found to be 100% resistant to co-amoxiclav, cefoxitin, ceftazidime, cefotaxime, cefuroxime, cefixime, ceftriaxone, cefpodoxime, gentamicin, amikacin and aztreonam. Less resistance was observed against cefepime (30.4%), sulbactam-cefoperazone (24.8%), piperacillin-tazobactam (10.6%), ciprofloxacin (20.5%) and meropenem (2.1%). All the isolates were found sensitive to imipenem. The patients harbored AmpC β-lactamases were on various interventions in which intravenous line was noted among (51.1%), naso-gastric tube (37.6%), ambu bag (8.5%), endotracheal tube (3.5%), ventilator (2.1%) and surgery (0.7%).Conclusion:Extensive use of invasive procedures and third generation cephalosporins should be restricted to avoid the emergence of AmpC beta-lactamases in neonates.

Exposing a β-Lactamase "Twist": the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase.

Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a β-lactamase profoundly affects β-lactam resistance.

Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa

Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the β-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB–oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD–OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms.

Structural and Kinetic Insights into the "Ceftazidimase" Behavior of the Extended-Spectrum β-Lactamase CTX-M-96.

Diversification of the CTX-M β-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 Å and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the β3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution.

Mutagenesis of the CTX-M-type ESBL-is MIC-guided treatment according to the new EUCAST recommendations a safe approach?

Spurred by the latest EUCAST and CLSI recommendation to adjust antibiotic therapy on the basis of MICs instead of resistance mechanisms, we aimed to investigate the ability of CTX-M-1 and CTX-M-14 to achieve ceftazidime resistance under selective conditions. We exposed Escherichia coli transconjugants bearing natural plasmids that express CTX-M-1, CTX-M-14 or CTX-M-15 to various selective culture conditions and tracked their growth and mutational frequencies. For selected mutants we analysed the sequences of the bla CTX-M genes, determined the altered MICs of cefotaxime, cefepime, ceftazidime and meropenem, and measured the efflux properties and the changes in transcriptional levels of bla CTX-M genes. The CTX-M-1- and CTX-M-14-bearing clones switched from ceftazidime-susceptible to ceftazidime-resistant phenotypes under selective conditions within 24 h. However, no mutations within the bla CTX-M genes were found, and the efflux was unlikely to be involved in the increased ceftazidime MICs. In CTX-M-1-bearing clones bla CTX-M-1 expression was 19-fold increased under ceftazidime-selective conditions but there was a high variance within the clones. Reasons for increased ceftazidime MICs of CTX-M-bearing clones remain unclear but might be the increased enzymatic activity or other intrachromosomal mutations. It can be speculated that different strategies to survive under selective conditions can be adopted by E. coli, thereby establishing an optimal mechanism with the lowest energy demand for each transconjugant. Based on our in vitro findings, we cannot fully recommend the use of ceftazidime, particularly in critically ill patients with infections due to ESBL producers, regardless of susceptibility to ceftazidime.

Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability

The spread of β-lactamases that hydrolyze penicillins, cephalosporins and carbapenems among Gram-negative bacteria has limited options for treating bacterial infections. Initially, Klebsiella pneumoniae carbapenemase-2 (KPC-2) emerged as a widespread carbapenem hydrolyzing β-lactamase that also hydrolyzes penicillins and cephalosporins but not cephamycins and ceftazidime. In recent years, single and double amino acid substitution variants of KPC-2 have emerged among clinical isolates that show increased resistance to ceftazidime. Because it confers multi-drug resistance, KPC β-lactamase is a threat to public health. In this study, the evolution of KPC-2 function was determined in nine clinically isolated variants by examining the effects of the substitutions on enzyme kinetic parameters, protein stability and antibiotic resistance profile. The results indicate that the amino acid substitutions associated with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime hydrolysis and a consequent increase in ceftazidime resistance. Single substitutions lead to modest increases in catalytic activity while the double mutants exhibit significantly increased ceftazidime hydrolysis and resistance levels. The P104R, V240G and H274Y substitutions in single and double mutant combinations lead to the largest increases in ceftazidime hydrolysis and resistance. Molecular modeling suggests that the P104R and H274Y mutations could facilitate ceftazidime hydrolysis through increased hydrogen bonding interactions with the substrate while the V240G substitution may enhance backbone flexibility so that larger substrates might be accommodated in the active site. Additionally, we observed a strong correlation between gain of catalytic function for ceftazidime hydrolysis and loss of enzyme stability, which is in agreement with the ‘stability-function tradeoff’ phenomenon. The high Tm of KPC-2 (66.5°C) provides an evolutionary advantage as compared to other class A enzymes such as TEM (51.5°C) and CTX-M (51°C) in that it can acquire multiple destabilizing substitutions without losing the ability to fold into a functional enzyme.

Epidemiology and emerging resistance in bacterial bloodstream infections in patients with hematologic malignancies

Background: The objective of this study was determine the frequency of bloodstream infections (BSIs) and the causative bacteria and their resistance patterns in patients with hematological malignancies (HMs) in a large tertiary care university hospital in Turkey over a 5-year period. Methods: A total of 2098 patients with HMs with 3703 neutropenic episodes were included. Patients were classified as high-risk (n = 843) and low-risk (n = 1255) groups and evaluated for frequency of BSIs, causative bacteria, and their resistance patterns. Results: The frequency of BSIs was 14.5%. The frequency of gram-negative BSIs in high-risk and low-risk groups was 10.7% and 5.4% (p < 0.001), respectively. The frequency of gram-positive BSIs in high-risk and low-risk groups was 7.0% and 3.9% (p < 0.001), respectively. Gram-negative bacteria predominated (52.6%), with Escherichia coli (17.3%) and Klebsiella spp. (11.0%) as the most frequent organisms. Coagulase-negative staphylococci (10.4%) and Corynebacterium spp. (6.3%) were the most common gram-positive bacteria (35.8%). The rate of extended-spectrum beta-lactamase (ESBL) production was 45% for E. coli and 58% for Klebsiella spp. Quinolone resistance was 58% for E. coli and 11% for Klebsiella spp.. The overall frequency of ceftazidime resistance in Pseudomonas aeruginosa was 28%, and 87% of Acinetobacter spp. were multidrug-resistant. Of Staphylococcus aureus isolates, 24.8% were resistant to methicillin. Conclusion: The dominating causes of BSIs in patients with HMs in our hospital are resistant gram-negative bacteria, which has made empirical antimicrobial choice a highly challenging issue in this patient population.

Activity of ceftazidime/avibactam against isogenic strains of Escherichia coli containing KPC and SHV β-lactamases with single amino acid substitutions in the Ω-loop.

The objective of this study was to explore the activity of ceftazidime and ceftazidime/avibactam against a collection of isogenic strains of Escherichia coli DH10B possessing SHV and KPC β-lactamases containing single amino acid substitutions in the Ω-loop (residues 164-179). Ceftazidime and ceftazidime/avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 β-lactamase variants that possessed elevated MICs of ceftazidime/avibactam were selected for further biochemical analyses. Avibactam restored susceptibility to ceftazidime for all Ω-loop variants of SHV-1 with MICs <8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/avibactam >8 mg/L. β-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 β-lactamases indicated that avibactam effectively inhibited these enzymes. Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in the Ω-loop were not susceptible to ceftazidime/avibactam (MICs >8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant β-lactamases that prevent avibactam from binding to and inhibiting the β-lactamase. As ceftazidime/avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important.

Influence of antimicrobial consumption on gram-negative bacteria in inpatients receiving antimicrobial resistance therapy from 2008-2013 at a tertiary hospital in Shanghai, China

Irrational use of antimicrobial agents is a major cause of increased antimicrobial resistance. Effective antibiotic stewardship strategies nationwide or in local health care settings are necessary to reduce antibiotic use and bacteria resistance. We evaluated the effectiveness of China's antimicrobial stewardship policy on antimicrobial use and applied time-series analysis methodology to determine the temporal relationship between antibiotic use and gram-negative bacteria resistance at Changhai Hospital from 2008-2013. Isolates investigated included Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Consumption of 7 restricted-use antibiotics was dramatically reduced. Resistance to ceftazidime in P aeruginosa and A baumannii and resistance to ciprofloxacin in P aeruginosa significantly decreased. By using cross-correlation analysis, associations between ciprofloxacin resistance in P aeruginosa and fluoroquinolones consumption (r = 0.48; lag = 0; P = .02), ceftazidime resistance in P aeruginosa and third-generation cephalosporins consumption (r = 0.54; lag = -1; P = .01) were identified. No substantial association between other pairs was found. Enhanced nationwide antimicrobial stewardship campaigns launched in 2011 have made great achievements in regard to antibiotic use but have had limited effects on the reversal of gram-negative bacteria resistance in health care settings. Sound infection prevention and control programs to reduce the transmission of resistant pathogens for hospitals in China are urgently needed.

In vitro susceptibility of fosfomycin against Extended-Spectrum β-lactamase -producing Escherichia coli isolated from urine specimens

Background: The production of extended-spectrum β-lactamases (ESBLs) brings challenge to the treatment of urinary infection caused by Escherichia coli with increasing resistance to common antibiotics. For the limited treatment option, fosfomycin as traditional antibiotic has been reevaluated. Yet there are scarce studies regarding the antibacterial activity of fosfomycin for urinary infection in China. With this regard, we investigated in vitro susceptibility of fosfomycin and other 7 agents against ESBLs-producing Escherichia coli isolated from urine specimens. Methods: The minimum inhibitory concentrations (MICs) of fosfomycin and other comparative agents including meropenem, piperacillin-tazobactam, amikacin, cefepime, cefotaxime, ceftazidime and levofloxacin were determined by agar dilution method recommended by the Clinical Laboratory Standards Institute (CLSI)2010. Results : Of the 100 strains isolated from urine, none was found resistant to meropenem .The relatively high susceptibility was found to piperacillin-tazobactam (94%), amikacin (85%), fosfomycin (82%) and cefepime (60%).The susceptibility rate of fosfomycin did not differ significantly between the cefotaxime-resistant(CTX-R) and cefotaxime- susceptible(CTX-S)group(80.1%vs84.6%,P=0.712), ceftazidime-resistant(CAZ-R) and ceftazidime-susceptible(CAZ-S)group(78.1%vs84.8%,P=0.552), levofloxacin-resistant (LVX-R) and levofloxacin-susceptible(LVX-S) group(85.4%vs62.5%,P=0.07) respectively. Conclusions: For the relatively high antibacterial activity in virto against ESBLs-producing Escherichia coli isolated from urine specimens and the non-influenced by the pattern of resistance to other antimicrobials, fosfomycin is of great clinical value in the treatment of uncomplicated urinary infection caused by ESBLs-producing Escherichia coli.

Characterization by phenotypic and genotypic methods of metallo-β-lactamase-producing Pseudomonas aeruginosa isolated from patients with cystic fibrosis.

Pseudomonas aeruginosa continues to be a predominant cause of infections with high intrinsic resistance to antibiotics, resulting in treatment failure. P. aeruginosa is the leading cause of respiratory infections among cystic fibrosis (CF) patients. Resistance to carbapenem antibiotics among P. aeruginosa has been reported. Thus, this study was undertaken to characterize the metallo-β-lactamase (MBL) production of P. aeruginosa by phenotypic and genotypic methods. A total of 572 sputum samples were collected from cystic fibrosis patients along with the patient demographic details in a questionnaire. In total, 217 P. aeruginosa isolates were collected and an antibiogram revealed that 159 (73.3%) and 141 (64.9%) of these colonies exhibited resistance to imipenem and meropenem, respectively. Ceftazidime and tobramycin resistance were both identified in 112 (51.6%) isolates, and resistance to piperacillin-tazobactam, gatifloxacin and netilmicin was detected in 96 (44.2%) respective samples. A total of 62 (28.6%) respective samples were resistant to cefoperazone, cefepime and ceftriaxone. The least antibiotic resistance was shown to amikacin and ceftizoxime with 51 (23.5%) and 32 (14.7%) respective colonies resistant to the antibiotics. The minimum inhibitory concentration (MIC) for imipenem revealed a reduction in the MIC values. MBL screening by the zone enhancement method using ceftazidime plus EDTA discs demonstrated that 63 (56.25%) of the colonies were positive for MBL. A total of 53 (84.1%) samples expressed blaVIM and 48 (76.1%) expressed blaIMP genes, as detected by duplex polymerase chain reaction. In conclusion, carbapenem resistance is of great clinical concern in cystic fibrosis patients with P. aeruginosa infection. Therefore, mandatory regular screening and monitoring the resistance in P. aeruginosa among CF patients is required.

Acinetobacter Peritoneal Dialysis Peritonitis: A Changing Landscape over Time

Background Acinetobacter species are assuming an increasingly important role in modern medicine, with their persistent presence in health-care settings and antibiotic resistance. However, clinical reports addressing this issue in patients with peritoneal dialysis (PD) peritonitis are rare.MethodsAll PD peritonitis episodes caused by Acinetobacter that occurred between 1985 and 2012 at a single centre were retrospectively reviewed. Clinical features, microbiological data, and outcomes were analysed, with stratifications based upon temporal periods (before and after 2000).Results Acinetobacter species were responsible for 26 PD peritonitis episodes (3.5% of all episodes) in 25 patients. A. baumannii was the most common pathogen (54%), followed by A. iwoffii (35%), with the former being predominant after 2000. Significantly more episodes resulted from breaks in exchange sterility after 2000, while those from exit site infections decreased (P = 0.01). The interval between the last and current peritonitis episodes lengthened significantly after 2000 (5 vs. 13.6 months; P = 0.05). All the isolates were susceptible to cefepime, fluoroquinolone, and aminoglycosides, with a low ceftazidime resistance rate (16%). Nearly half of the patients (46%) required hospitalisation for their Acinetobacter PD-associated peritonitis, and 27% required an antibiotic switch. The overall outcome was fair, with no mortality and a 12% technique failure rate, without obvious interval differences.ConclusionsThe temporal change in the microbiology and origin of Acinetobacter PD-associated peritonitis in our cohort suggested an important evolutional trend. Appropriate measures, including technique re-education and sterility maintenance, should be taken to decrease the Acinetobacter peritonitis incidence in PD patients.

Deletion mutations conferring substrate spectrum extension in the class A β-lactamase.

We describe four new deletion mutations in a class A β-lactamase PenA in Burkholderia thailandensis, each conferring an extended substrate spectrum. Single-amino-acid deletions T171del, I173del, and P174del and a two-amino-acid deletion, R165_T167delinsP, occurred in the omega loop, increasing the flexibility of the binding cavity. This rare collection of mutations has significance, allowing exploration of the diverse evolutionary trajectories of β-lactamases and as potential future mutations conferring high-level ceftazidime resistance on isolates from clinical settings, compared with amino acid substitution mutations.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients.

To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance.

Prevalence of nosocomial infections and anti-infective therapy in Benin: results of the first nationwide survey in 2012.

BackgroundData on nosocomial infections in hospitals in low-income countries are scarce and often inconsistent. The objectives of this study were to estimate the prevalence of nosocomial infections and antimicrobial drug use in Benin hospitals.MethodsAll hospitals were invited to participate in the first national point prevalence study conducted between 10–26 October 2012 using the protocol developed by the “Hospitals in Europe Link for Infection Control through Surveillance” (HELICS) project. Infection prevalence rates and the proportion of infected patients and exposure to antimicrobials were assessed.ResultsOverall, 87% (39/45) of hospitals participated. Of 3130 inpatients surveyed, 972 nosocomial infections were identified among 597 patients, representing an overall prevalence of infected patients of 19.1%. The most frequent infections were related to the urinary tract (48.2%), vascular catheter use (34.7%), and surgical site (24.7%). 64.6% of patients surveyed were treated with antibiotics, including a significant proportion (30%) of non-infected patients and a high proportion of self-medication (40.8%). Resistance of leading nosocomial pathogens to antimicrobials included methicillin-resistance (52.5%) among Staphylococcus aureus, vancomycin resistance among enterococci (67.5%), cefotaxime resistance among Escherichia coli (67.6%), and ceftazidime resistance among Acinetobacter baumannii (100%) and Pseudomonas aeruginosa (68.2%).ConclusionsBenin has high nosocomial infection rates and calls for the implementation of new national infection control policies. Patient safety education and training of all individuals involved in healthcare delivery will be critical to highlight awareness of the burden of disease. The high use of antimicrobials needs to be addressed, particularly their indiscriminate use in non-infected patients.

Rapid Burkholderia pseudomallei identification and antibiotic resistance determination by bacteriophage amplification and MALDI-TOF MS.

Phage amplification detected by MALDI-TOF MS was investigated for rapid and simultaneous Burkholderia pseudomallei identification and ceftazidime resistance determination. B. pseudomallei ceftazidime susceptible and resistant ΔpurM mutant strains Bp82 and Bp82.3 were infected with broadly targeting B. pseudomallei phage ϕX216 and production of the m/z 37.6 kDa phage capsid protein observed by MALDI-TOF MS over the course of 3 h infections. This allowed for repoducible phage-based bacterial ID within 2 h of the onset of infection. MALDI-TOF MS-measured time to detection correlated with in silico modeling, which predicted an approximate 2 h detection time. Ceftazidime susceptible strain Bp82, while detectable in the absence of the drug, owing to the reliance of phage amplification on a viable host, was not detectable when 10 μg/mL ceftazidime was added at the onset of infection. In contrast, resistant strain Bp82.3 was detected in the same 2 h timeframe both with and without the addition of ceftazidime.

Molecular epidemiology of Pseudomonas aeruginosa clinical isolates from Korea producing β-lactamases with extended-spectrum activity

This study was performed to investigate the prevalence and molecular epidemiology of Pseudomonas aeruginosa isolates from Korea that produce enzymes with extended-spectrum (ES) activity to β-lactams. A total of 205 non-duplicate P. aeruginosa clinical isolates were collected from 18 university hospitals in Korea. PCR and sequencing experiments were performed to identify genes encoding β-lactamases. PCR mapping and sequencing of the regions surrounding the β-lactamase genes were performed. Multilocus sequence typing experiments were performed. The most common sequence type (ST) was ST235 (n = 96), and 2 single-locus variants of ST235, ST1015 (n = 1) and ST1162 (n = 1), were also identified. These 3 STs were grouped as a clonal complex (CC), CC235. The remaining 107 isolates were identified as 59 different STs. Isolates belonging to CC235 showed higher rates of non-susceptibility to imipenem (85.4% versus 47.7%) and meropenem (92.7% versus 52.3%) compared to non-CC235 isolates. All the metallo-β-lactamase (MBL)–producing isolates were identified as CC235, except for 1 ST591. Genes encoding OXA-17 and OXA-142 were detected in 1 isolate and 4 isolates of CC235, respectively; while the blaSHV-12 gene was detected in 4 non-CC235 isolates. Class A and D β-lactamases with ES activity play a role in acquiring ceftazidime resistance in P. aeruginosa in Korea. Production of IMP-6 and VIM-2 MBLs is the main mechanisms in acquiring resistance to ceftazidime and carbapenems in P. aeruginosa isolates in Korea. Clonal spread of P. aeruginosa CC235 may be an important conduit for the dissemination of MBL genes in Korea.

Outbreak of PER-1 and diversity of β-lactamases among ceftazidime-resistant Pseudomonas aeruginosa clinical isolates

A growing number of β-lactamases have been reported in Pseudomonas aeruginosa clinical isolates. The aim of this study was to investigate the diversity of β-lactamases in the collection of 51 ceftazidime-resistant P. aeruginosa clinical isolates in four hospitals of southern China. Among these isolates, variable degrees of resistance to other β-lactam and non-β-lactam agents were observed. Pulsed-field gel electrophoresis (PFGE) revealed a high degree of clonality with five main genotypes. Of the 51 isolates tested, 35 (68.6%) were identified as extended-spectrum β-lactamase (ESBL) producers, with 35 producing PER-1, 1 CTX-M-3, 7 CTX-M-15 and 1 CTX-M-14. Most (82.9%, 29/35) PER-1-producing isolates were collected from two hospitals between January and April in 2008 and belonged to the same PFGE pattern (pattern B) with similar antibiogram and β-lactamase profiles, which suggested an outbreak of this clone at the time. The prevalence of CTX-M-type ESBL (17.6%, 9/51) was unexpectedly high. One isolate was identified as producing VIM-2. Furthermore, we also reported an occurrence of a novel OXA-10 variant, OXA-246, in 14 P. aeruginosa isolates. In addition, AmpC overproduction was found to be the β-lactamase-mediated mechanism responsible for ceftazidime resistance in 6 isolates (11.8%). Our results revealed an overall diversity of β-lactamases and outbreak of a PER-1-producing clone among ceftazidime-resistant P. aeruginosa in southern China.

Rectal Colonization with Extended Spectrum β-Lactamase producing Enterobacteriacieae in Surgical Patients in a Tertiary Hospital in Calabar, Nigeria.

Aim: Extended spectrum β-lactamases are transferable plasmid mediated resistance mechanisms found mainly among Enterobacteriaceae which confer resistance to numerous β-lactam antibiotics. The aim of the study was to determine the rate of rectal colonization of surgical patients with Escherichia coli carrying extended spectrum β-lactamases. Subjects and methods: We collected and cultured rectal swabs from 192 surgical patients in the University of Calabar Teaching Hospital, Calabar, Nigeria. Escherichia coli isolates were identified and antibiotics susceptibility performed using Modified Kirby-Bauer disc diffusion technique. Isolates showing zones of inhibition to third generation cephalosporins below Clinical and Laboratory Standard Institute recommended break-points were screened for ESBL-production by the double-disk synergy method. Results: A total of 67.7%(130) of the patients received prophylactic antibiotics which was continued empirically for ≥5days after surgery. Ceftriaxone was the most empirically prescribed antibiotic used with metronidazole in 63.1%(82/130) and alone in 7.7%(10/130) of patients. Ceftriaxone resistance was observed in 39.6%(76/192) of the rectal E. coli isolates with 3.1%(6/192) showing intermediate susceptibility. Ceftazidime resistance was observed in 40.6%(78/192) with 4.2%(8/192) showing intermediate susceptibility. ESBL mediated resistance was observed in 27.1%(52/192) of isolates. ESBL positive isolates were more likely to be resistant to Gentamicin, Ciprofloxacin and Co-trimoxazole than negative isolates (P<0.001). All isolates were susceptible to Meropenem. Younger patients were more colonized with a preponderance in patients less than 9 years of age (Pearson's correlation = -0.196;P=0.006). Conclusion: Infection may occur in colonized surgical patients leading to increased morbidity. Pre-surgical screening to identify colonized patients will be beneficial in the early selection of appropriate therapy and management of outbreaks while functional hospital antibiotic stewardship programmes and strict enforcement of national antibiotic regulations will curtail their spread.

Antimicrobial susceptibility and genetic characterisation of Burkholderia pseudomallei isolated from Malaysian patients.

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B).