Evolvability as a function of purifying selection in TEM-1 ß-lactamase.

Evolvability­the capacity to generate beneficial heritable variation­is a central property of biological systems. However, its origins and modulation by environmental factors have not been examined systematically. Here, we analyze the fitness effects of all single mutations in TEM-1 ß-lactamase (4,997 variants) under selection for the wild-type function (ampicillin resistance) and for a new function (cefotaxime resistance). Tolerance to mutation in this enzyme is bimodal and dependent on the strength of purifying selection in vivo, a result that derives from a steep non-linear ampicillin-dependent relationship between biochemical activity and fitness. Interestingly, cefotaxime resistance emerges from mutations that are neutral at low levels of ampicillin but deleterious at high levels; thus the capacity to evolve new function also depends on the strength of selection. The key property controlling evolvability is an excess of enzymatic activity relative to the strength of selection, suggesting that fluctuating environments might select for high-activity enzymes.

Ultrasensitive Electrochemiluminescence Biosensor to Detect Ampicillin Resistance Gene (ARGAMP) Based on a Novel Near-Infrared Ruthenium Carbene Complex/TPrA/PEI Ternary ECL System.

The establishment of rapid target identification and analysis methods for antibiotic resistance genes (ARGs) is urgently needed. In this study, we unprecedently designed a target-catalyzed hairpin assembly (CHA) electrochemiluminescent (ECL) biosensor for the ultrasensitive detection of ampicillin resistance genes (ARGAMP) based on a novel, efficient near-infrared ruthenium carbene complex/TPrA/PEI ternary ECL system with low oxidation potential. The ternary NIR-ECL system illustrated in this work displayed double ECL intensity in comparison with their corresponding traditional binary ECL system. The as-prepared ECL biosensor illustrated in this work demonstrates highly selective and sensitive determination of ARGAMP from 1 fM to 1 nM and a low detection limit of 0.23 fM. Importantly, it also exhibits good accuracy and stabilities to identify ARGAMP in plasmid and bacterial genome DNA, which demonstrates its excellent reliability and great potential in detecting ARGAMP in real environmental samples.

Synthesis of a tetralone derivative of ampicillin to control ampicillin-resistant Staphylococcusaureus.

Due to the rapid emergence of antibiotic resistant new bacterial strains and new infections, there is an urgent need for novel or newly modified and efficient alternatives of treatment. However, conventional antibiotics are still used in therapeutic settings but their efficacy is uncertain due to the rapid evolution of drug resistance. In the present study, we have synthesized a new derivative of conventional antibiotic ampicillin using SN2-type substitution reaction. NMR and mass analysis of the newly synthesized derivative of ampicillin confirmed it as ampicillin-bromo-methoxy-tetralone (ABMT). Importantly, ABMT is revealed to have efficient activity against Staphylococcus aureus (S. aureus) with a MIC value of 32 µg ml-1 while ampicillin was not effective, even at 64 µg ml-1 of concentration. Electron microscopy results confirmed the membrane-specific killing of S. aureus at 1 h of treatment. Additionally, molecular docking analysis revealed a strong binding affinity of ABMT with ß-lactamase via the formation of a closed compact bridge. Our findings, avail a new derivative of ampicillin that could be a potential alternative to fight ampicillin-resistant bacteria possibly by neutralizing the ß-lactamase action.

Resistance mechanism of Escherichia coli strains with different ampicillin resistance levels.

Antibiotic resistance is an important problem that threatens medical treatment. Differences in the resistance levels of microorganisms cause great difficulties in understanding the mechanisms of antibiotic resistance. Therefore, the molecular reasons underlying the differences in the level of antibiotic resistance need to be clarified. For this purpose, genomic and transcriptomic analyses were performed on three Escherichia coli strains with varying degrees of adaptive resistance to ampicillin. Whole-genome sequencing of strains with different levels of resistance detected five mutations in strains with 10-fold resistance and two additional mutations in strains with 95-fold resistance. Overall, three of the seven mutations occurred as a single base change, while the other four occurred as insertions or deletions. While it was thought that 10-fold resistance was achieved by the effect of mutations in the ftsI, marAR, and rpoC genes, it was found that 95-fold resistance was achieved by the synergistic effect of five mutations and the ampC mutation. In addition, when the general transcriptomic profiles were examined, it was found that similar transcriptomic responses were elicited in strains with different levels of resistance. This study will improve our view of resistance mechanisms in bacteria with different levels of resistance and provide the basis for our understanding of the molecular mechanism of antibiotic resistance in ampicillin-resistant E. coli strains. KEY POINTS: •The mutation of the ampC promoter may act synergistically with other mutations and lead to higher resistance. •Similar transcriptomic responses to ampicillin are induced in strains with different levels of resistance. •Low antibiotic concentrations are the steps that allow rapid achievement of high antibiotic resistance.

A critical issue on microbiological cut-off value of ampicillin resistance in Lactiplantibacillus plantarum.

AIM: Comprehensive evaluation of antibiotic susceptibility patterns in Lactiplantibacillus plantarum strains isolated from grape marc, based on genomic and phenotypic assessment. METHODS AND RESULTS: We assessed the antibiotic resistance-susceptibility patterns of 20 L. plantarum strains for 16 antibiotics. Genomes of relevant strains were sequenced for in silico assessment and comparative genomic analysis. Results showed high MIC values for spectinomycin, vancomycin, and carbenicillin, indicating natural resistance to these antibiotics. Besides, these strains revealed MIC values for ampicillin higher than previously established by the EFSA, indicating the possible presence of acquired resistance genes in the genomes. However, genomic analysis by complete genome sequencing did not reveal presence of ampicillin resistance genes. CONCLUSION: Comparative genomic analysis between our strains and other L. plantarum genomes present in the literature showed several substantial genomic differences, and suggested the need to adjust the cut-off value for ampicillin in L. plantarum. However, further sequence analysis will reveal how these strains have acquired antibiotic resistance.

A neglected risk of nanoplastics as revealed by the promoted transformation of plasmid-borne ampicillin resistance gene by Escherichia coli.

Plastic pollution and antibiotic resistance are two emerging environmental and human health crises today. Although it was revealed that microplastics can serve as vectors for the dissemination of antibiotic resistance, it is still unclear how the nanoplastics influence the horizontal transfer of antibiotic resistance genes (ARGs). Herein, we firstly compared the effect of polystyrene (PS) micro/nanoplastics on the transformation of plasmid-borne ARG, using a transformation model consisting of plasmid pUC19 (ampR ) and Escherichia coli DH5α (recipient). Due to its size effect, PS nanoplastics (10-500 mg/L) significantly enhanced the transformation efficiency (2.8-5.4 folds) and frequency (3.2-8.4 folds) of exogenous ampR into E. coli, while PS microplastics exerted no influence. The detailed mechanisms were found that nanoplastics induced reactive oxygen species (ROS) overproduction, activated SOS response, increased cell membrane permeability and changed the secretion systems, thereby facilitating the uptake of exogenous DNA by bacteria. Moreover, the co-presences of nanoplastics with humic acid or Fe3+ relieved to some extent, but did not completely alleviate the promoting effect of nanoplastics on plasmid transformation. Our findings suggest that the risk of nanoplastics on promoting the dissemination of antibiotic resistance should not be neglected, and further studies are needed to investigate such risk in complex environments.

Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

Metabolic phenotyping of acquired ampicillin resistance using microbial volatiles from Escherichia coli cultures.

AIMS: This study sought to assess the volatile organic compound (VOC) profiles of ampicillin-resistant and -susceptible Escherichia coli to evaluate whether VOC analysis may be utilized to identify resistant phenotypes. METHODS AND RESULTS: An E. coli BL21 (DE3) strain and its pET16b plasmid transformed ampicillin-resistant counterpart were cultured for 6 h in drug-free, low- and high-concentrations of ampicillin. Headspace analysis was undertaken using thermal desorption-gas chromatography-mass spectrometry. Results revealed distinct VOC profiles with ampicillin-resistant bacteria distinguishable from their susceptible counterparts using as few as six compounds. A minimum of 30 compounds (fold change >2, p ≤ 0.05) were differentially expressed between the strains across all set-ups. Furthermore, three compounds (indole, acetoin and 3-methyl-1-butanol) were observed to be significantly more abundant (fold change >2, p ≤ 0.05) in the resistant strain compared to the susceptible strain both in the presence and in the absence of drug stress. CONCLUSIONS: Results indicate that E. coli with acquired ampicillin resistance exhibit an altered VOC profile compared to their susceptible counterpart both in the presence and in the absence of antibiotic stress. This suggests that there are fundamental differences between the metabolisms of ampicillin-resistant and -susceptible E. coli which may be detected by means of VOC analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that VOC profiles may be utilized to differentiate between resistant and susceptible bacteria using just six compounds. Consequently, the development of machine-learning models using VOC signatures shows considerable diagnostic applicability for the rapid and accurate detection of antimicrobial resistance.

Haemophilus influenzae one day in Denmark: prevalence, circulating clones, and dismal resistance to aminopenicillins.

Haemophilus influenzae is a common cause of mucosal infections that warrants accurate surveillance. We aimed to assess the prevalence of the species in clinical specimens, and characterise population structure and resistance to aminopenicillins by whole genome sequencing.We assessed the point prevalence by entering the database records of 1 day in Denmark and examined the genome sequences of nationwide, collected isolates from the same day. The prevalence of H. influenzae in clinical samples on the 10th of January 2018 was 1.78 per 100,000 person-days (all samples), and 2.47 per 1000 hospital bed-days (hospital samples). Of 2009 bacteria deemed clinically relevant and collected in a concerted action by the Danish departments of clinical microbiology, 62 (3.1%) were H. influenzae. All 62 isolates belonged to phylogenetic group I and were unencapsulated. Three strains from separate Danish regions had identical core genome sequences, but a small number of intergenic mutations testified to circulating clones, rather than individual cases of patient-to-patient transmission. The TEM-1 ß-lactamase gene was present in 24 strains, while 13 strains were genetically categorised as ampicillin-resistant due to substitutions in penicillin-binding protein 3; shared patterns of amino acid substitutions in unrelated strains indicated putative lateral transfer of chromosomal resistance. Circulating clones of H. influenzae are frequent, and host factors, rather than direct transmission of epidemic strains, may be the primary cause of infection. The bleak presence of ampicillin resistance revealed by sequencing of point prevalence strains underscores the necessity for close examination of testing methods.

Antibacterial and antibiofilm effects of flufenamic acid against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most serious surgery complications, and their prevention is of utmost importance. Flufenamic acid is a non-steroid anti-inflammatory drug approved for clinical use to relieve inflammation and pain in rheumatoid arthritis patients. In this study, we explored the antibacterial efficacy of flufenamic acid and the mechanisms underlying this effect. By using minimal inhibitory concentration (MIC), time-kill, resistance induction assays, and the antibiotic synergy test, we demonstrated that flufenamic acid inhibited the growth of methicillin-resistant staphylococci and did not induce resistance when it was used at the MIC. Furthermore, flufenamic acid acted synergistically with the beta-lactam antibiotic oxacillin and did not show significant toxicity toward mammalian cells. The biofilm inhibition assay revealed that flufenamic acid could prevent biofilm formation on medical implants and destroy the ultrastructure of the bacterial cell wall. RNA sequencing and quantitative RT-PCR indicated that flufenamic acid inhibited the expression of genes associated with peptidoglycan biosynthesis, beta-lactam resistance, quorum sensing, and biofilm formation. Furthermore, flufenamic acid efficiently ameliorated a local infection caused by MRSA in mice. In conclusion, flufenamic acid may be a potent therapeutic compound against MRSA infections and a promising candidate for antimicrobial coating of implants and surgical devices.

Epidemiology of Haemophilus influenzae in the Republic of Ireland, 2010-2018.

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. ß-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected ß-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

Ampicillin-mediated functionalized gold nanoparticles against ampicillin-resistant bacteria: strategy, preparation and interaction studies.

Antibiotic resistance is a highly challenging concern of infectious diseases, and it requires a rational approach to overcome. Through this work, we have synthesized ampicillin-capped gold nanoparticles (Amp-Au NPs) and studied its interaction with bacterial cells. In this process of synthesis, the primary amine group of ampicillin acts as both reducing as well as capping agent. In addition to synthesized gold nanoparticles, the ß-lactam ring remains free to interact with bacteria. This approach not only utilizes the maximum efficiency of nanoparticles and antibiotics towards ampicillin sensitive bacterial cells but also proves to be effective against ampicillin resistance bacteria. Our results illustrate that the optimized system of Amp-Au NPs was formulated by taking 1.25 mM ampicillin and 10-2 of gold ions concentration. UV-vis spectrum of gold nanoparticles and the presence of ampicillin were recorded at around 540 nm and 259 nm, respectively. Microscopic images indicate that particles are nearly spherical and are in size range between 25 and 50 nm. Moreover, formulated Amp-Au NPs show successful accumulation onto the surface of the bacterial cell as a result of which pores were formed into the bacterial membrane. The entry of nanoparticles into bacterial cells was validated through both atomic force microscopy and fluorescent microscopy. The adhesive properties of this coating material and its stability in various pH, i.e. pH 3, pH 7 and pH 10 conditions, could make them a good candidate in the prevention of biofilm formation. Amp-Au NPs show promising antimicrobial activity against ampicillin resistance Escherichia coli bacteria. Furthermore, antimicrobial studies indicate that the efficacy of Amp-Au NPs increased against both ampicillin sensitive and ampicillin resistance bacteria up to sixteen folds and four folds respectively.

Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine.

Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 ß-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.

The non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli.

OBJECTIVES: The non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli. METHODS: Bacterial DNA was extracted by boiling method and then amplified using polymerase chain reaction (PCR) with two different primer sets. Recombinant pET28a/rprA-sense and -antisense plasmids were separately transferred into the competent E. coli BL21 (DE3) by chemical methods using heat shock. The expression was analyzed at the RNA level using Semi quantitative RT PCR. The turbidity difference between the bacteria was checked by Broth Dilution method. RESULTS: The statistical analysis showed that the turbidity difference between the up regulated and control bacteria is significant (p value < 0.0001). The ANOVA test also showed the significant difference between the down regulated and control bacteria (p value < 0.0001). CONCLUSION: Considering this mechanism, there are some reports indicating the role of rprA in antibiotic resistance. However, the role of rprA in ampicillin resistance is remained to be unknown. The aim of this study was to analyze the up regulation and down regulation of rprA and check their effects on ampicillin resistance in Escherichia coli. It was found that the up regulation and down regulation of rprA can lead into more antibiotics resistance and susceptibility, respectively. Our results showed the potential role of rprA expression in the response to ampicillin stress in E. coli.

Bacterial profile and antibacterial susceptibility of otitis media among pediatric patients in Hawassa, Southern Ethiopia: cross-sectional study.

BACKGROUND: Otitis Media (OM) is the most common disease of childhood. Twenty thousand people die each year from otitis media. It is an important cause of preventable hearing loss, affects children's intellectual performance and language development. There are very small numbers of studies done in Ethiopia concerning this topic. This study aimed to identify bacterial pathogens related to ear infection and to assess antibacterial susceptibility of isolated organisms. METHOD: A cross-sectional study was conducted on 152 children from April 2018 to July 2018 at selected health facilities in Hawassa city, SNNPR, Ethiopia. All pediatric patients having ear discharge were included. Convenient sampling technique was used to collect clinical and demographic data using standard questionnaires after child care-takers signed the consent. Ear discharge specimens were collected using a sterile swab, and transported using Amies transport media to Hawassa University Comprehensive Specialized Hospital laboratory. Bacterial isolates were characterized based on colony appearance, Gram reaction, culture characteristics, and biochemical tests after inoculating on appropriate culture media. Antibacterial susceptibility testing was performed using the disc diffusion method according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Among 152 children included, 115(75.6%) of them demonstrated pathogenic bacterial growth. Staphylococcus aureus 41(27%) was the most frequently isolated pathogen, followed by Proteus mirabilis 19 (12.5%). Of the total isolates, 11.2 and 7.3% were resistant to gentamicin and ciprofloxacin respectively. Over three-fourth (85.2%) of the isolates were resistant to ampicillin. More than two-third of the isolates were resistant to both penicillin (71.4%) and trimethoprim-sulphamethoxazole (72.0%). CONCLUSIONS: S. aureus is the most commonly isolated bacterial pathogen from ear discharge among children. Even though gentamicin is a parenteral drug and ciprofloxacin is rarely used in children due to concerns of bone/joint effects, these two drugs were highly effective antibiotics and thus should be considered in treating children with otitis media since most organisms were resistance or poor response to first line drugsHigh level of antibiotic resistance was observed so antimicrobial susceptibility test is needed before prescribing drugs for treatment of OM.

Multiclonal Expansion and High Prevalence of ß-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones.

ß-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were ß-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

Distribution of putative virulence markers in Enterococcus faecium: towards a safety profile review.

Objectives: The criteria for identification of Enterococcus faecium (Efm) with the ability to cause human infections are currently being debated by the European Food Safety Authority (EFSA). Strains that have an MIC of ampicillin of ≤ 2 mg/L and lack IS16/esp/hyl genes should be regarded as safe for use as feed additives in animal nutrition, despite the lack of knowledge about putative virulence marker (PVM) distribution in community Efm. We analysed the distribution of major PVM and ampicillin phenotypes in large Efm collections to investigate further the safety of strains from a public health perspective. Methods: Thirty-three PVM were assessed by PCR/sequencing among clonally disparate Efm (n = 328; 1986-2015) from different origins. We analysed ampicillin susceptibility (Etest/broth microdilution) according to EUCAST guidelines, clonal relationship (MLST) and genomic location of PVM (S1-PFGE/hybridization). Results: Infection-derived Efm were more enriched in PVM and the increase in ampicillin MIC was positively correlated with an enrichment in different PVM. PVM coding for surface (esp/sgrA/ecbA/complete acm) and pili proteins, or others enhancing colonization (hyl/ptsD/orf1481) or plasticity (IS16), were strongly associated with clinical Efm (mostly clade A1), but also observed in clades A2/B at different rates. ptsD was a good marker of ampicillin-resistant Efm. ptsD, IS16, orf1481, sgrA and hospital variants of complete pili gene clusters are proposed as markers to assess the safety of Efm strains. Conclusions: Our study expands on the distribution of PVM in diverse Efm lineages and demonstrates the enrichment in infection-derived strains of PVM not previously included in EFSA's list of Efm safety criteria. The evidence of relevant Efm infection markers can impact the risk assessment of Efm strains in different public health contexts.

Enterococcal bacteraemia: predictive and prognostic risk factors for ampicillin resistance.

To identify the predictive and prognostic factors associated with ampicillin-resistant enterococcal bacteraemia, we retrospectively reviewed demographic, microbiological and clinical data of patients attending the Kyoto University Hospital, Japan, between 2009 and 2015. Logistic regression and Cox regression analyses were performed to determine the predictive and prognostic factors, respectively. In total, 235 episodes of enterococcal bacteraemia were identified. As ampicillin susceptibility was uniform for Enterococcus faecalis isolates and almost all ampicillin-resistant isolates were E. faecium, bacteraemia due to these species was investigated separately. E. faecalis and E. faecium accounted for 41.7% (98/235) and 48.1% (113/235) of the isolates, respectively and 91.2% of all E. faecium were ampicillin resistant. Nosocomial E. faecium bacteraemia acquisition (odds ratio (OR), 13.6; 95% confidence intervals, 3.16-58.3) was associated with ampicillin-resistant isolates. Bacteraemia from an unknown source (hazard ratio (HR), 2.91; 95% CI 1.36-6.21) and an increased Pitt bacteraemia score (PBS) (HR, 1.36; 95% CI 1.21-1.52) were associated with 30-day mortality in E. faecium infections. Likewise, bacteraemia from an unknown source (HR, 4.17; 95% CI 1.25-13.9) and increased PBS (HR, 1.27; 95% CI 1.09-1.48) were associated with 30-day mortality in patients with E. faecalis bacteraemia. The empirical therapeutic administration of glycopeptides is recommended for patients with bacteraemia from an unknown source in whom severe E. faecium bacteraemia is suspected.

Molecular diagnosis and characterization of a culture-negative mycotic aneurysm due to ST54 Haemophilus influenzae type b with PBP 3 alterations.

Mycotic aneurysm is a rare but life-threatening disease that warrants an integrated therapeutic approach involving surgical intervention and prolonged antibiotic use. However, the causative organisms are often unidentified because antibiotics started empirically render blood and tissue cultures negative. Molecular diagnosis has been reported to be useful in such culture-negative cases. We report a case of a culture-negative mycotic aortic aneurysm due to Haemophilus influenzae, diagnosed by direct 16S rRNA polymerase chain reaction (PCR) and sequencing of the resected aneurysm tissue. PCR for serotype revealed type b, and PCR and sequencing of the ftsI gene revealed alterations in penicillin-binding protein 3, suggesting resistance to ampicillin. Multilocus sequence typing demonstrated that the isolate belonged to sequence type 54.

Imidazole decreases the ampicillin resistance of an Escherichia coli strain isolated from a cow with mastitis by inhibiting the function of autoinducer 2.

Extended-spectrum ß-lactamase-positive Escherichia coli is an important causative agent of mastitis in dairy cows that results in reduced milk production and quality, and is responsible for severe economic losses in the dairy industry worldwide. The quorum sensing signaling molecule autoinducer 2 (AI-2) is produced by many species of gram-negative and gram-positive bacteria, and might be a universal language for intraspecies and interspecies communication. Our previous work confirmed that exogenous AI-2 increases the antibiotic resistance of extended-spectrum ß-lactamase-positive E. coli to the ß-lactam group of antibiotics by upregulating the expression of the TEM-type ß-lactamase. In addition, this regulation relies on the function of the intracellular AI-2 receptor LsrR. In the present work, we reported that exogenous imidazole, a furan carbocyclic analog of AI-2, decreases the antibiotic resistance of a clinical E. coli strain to ß-lactam antibiotics by inhibiting the function of AI-2.