MICs Of Cefotaxime Research Articles

Uncovering the Important Genetic Factors for Growth during Cefotaxime-Gentamicin Combination Treatment in blaCTX-M-1 Encoding Escherichia coli.

Due to the rapid spread of CTX-M type ESBLs, the rate of resistance to third-generation cephalosporin has increased among Gram-negative bacteria, especially in Escherichia coli, and there is a need to find ways to re-sensitize ESBL E. coli to cephalosporin treatment. A previous study showed that genes involved in protein synthesis were significantly up-regulated in the presence of subinhibitory concentration of cefotaxime (CTX) in a CTX-M-1-producing E. coli. In this study, the interaction between CTX and gentamicin (GEN), targeting protein synthesis, was evaluated in MG1655/pTF2, and the MIC of CTX was strongly reduced (128-fold) in the presence of this combnation therapy. Since the underlying mechanism behind this synergy is not known, we constructed a saturated transposon mutant library in MG1655/pTF2::blaCTX-M-1 containing 315,925 unique transposon insertions to measure mutant depletion upon exposure to CTX, GEN, and combination treatment of CTX and GEN by Transposon Directed Insertion-site Sequencing (TraDIS). We identified 57 genes that were depleted (log2FC ≤ -2 and with q.value ≤ 0.01) during exposure to CTX, 18 for GEN, and 31 for combination treatment of CTX and GEN. For validation, we deleted eight genes that were either uniquely identified in combination treatment, overlapped with monotherapy of GEN, or were shared between combination treatment and monotherapy with CTX and GEN. Of these genes, we found that the inactivation of dnaK, mnmA, rsgA, and ybeD increased the efficacy of both CTX and GEN treatment, the inactivation of cpxR and yafN increased the efficacy of only CTX, and the inactivation of mnmA, rsgA, and ybeD resulted in increased synergy between CTX and GEN. Thus, the study points to putative targets for helper drugs that can restore susceptibility to these important drugs, and it indicates that genes involved in protein synthesis are essential for the synergy between these two drugs. In summary, the study identified mutants that sensitize ESBL-producing E. coli to CTX and a combination of CTX and GEN, and it increased our understanding of the mechanism behind synergy between β-lactam and aminoglycoside drugs. This forms a framework for developing new strategies to combat infections caused by resistant bacteria.

Combined Antibacterial activity of Eucalyptol, γ-terpinene, p-cymol and punicalagin with Cefotaxime against Methicillin (Oxacillin) Resistant Staphylococcus aureus Isolate

Resistance to antibiotics is a significant public health issue in preventing infectious diseases. This study was designed to evaluate the antibacterial activity of eucalyptol, γ-terpinene, p-cymol and punicalagin against Methicillin (Oxacillin) resistant strains. The synergistic effect of these compounds with cefotaxime against Staphylococcus aureus was investigated. The lowest MIC value observed was 0.08mg/mL for punicalagin against S. aureus, followed by the MIC values of punicalagin against E. aerogenes, E. coli and K. pneumoniae (0.16 - 0.63mg/mL). The activity of p-cymol against S. aureus is ranked second, as indicated by the MIC value (0.63mg/mL). The results of the combination study showed that the maximum Increase in Folding Area (IFA) was reported when punicalagin was combined with cefoxitin, cefotaxime, oxacillin, and piperacillin. An effective IFA was observed when p-cymol was tested with piperacillin, cefixime, cefotaxime, oxacillin, and cefoxitin. Based on the checkerboard assay, the MIC of cefotaxime decreased from 60 to 7.5µg/mL when combined with punicalagin indicating a significant synergistic effect between cefotaxime and punicalagin against S. aureus. In conclusion, punicalagin improved S. aureus susceptibility to cefotaxime, suggesting that using these two medicines together can reverse beta-lactam resistance in methicillin (oxacillin) resistant S. aureus. Further works are required to generalize this result and then it may be useful for treating diseases caused by methicillin (oxacillin) resistant strains.

Resistance to fosfomycin is increasing and is significantly associated with extended-spectrum β-lactamase-production in urinary isolates of Escherichia coli

Fosfomycin has become a therapeutic option in urinary tract infections. Our objective was to evaluate the in vitro activity of fosfomycin against Escherichia coli isolated from urine samples in 2013, 2018 and 2021. We also determined a putative association between fosfomycin resistance and extended-spectrum β-lactamases (ESBL) production. Fosfomycin activity was evaluated against 7367, 8128 and 5072 Escherichia coli urinary isolates in 2013, 2018 and 2021, respectively. We compare the prevalence of fosfomycin-resistant strains among the ESBL- and non-ESBL-producing isolates. MICs of fosfomycin, cefotaxime, and cefotaxime-clavulanate were determined by a microdilution method. 302 ESBL-producers were selected to determine MICs of fosfomycin by agar dilution and genes encoding ESBLs were detected by PCR. Among the total of ESBL-producing strains, 14.3%, 20.8% and 20% were resistant to fosfomycin in 2013, 2018 and 2021, respectively, whereas fosfomycin resistance in non-ESBL producers was 3.5%, 4.05% and 5.53% for each year (P ≤ 0.001). In the 302 selected ESBL-producing isolates, CTX-M was the main ESBL (228 isolates), being 50.7% CTX-M-15. Resistance to fosfomycin among these ESBL-producing strains was associated (P = 0.049) with isolates that produced the CTX-M type. Our data show that fosfomycin resistance is increasing in Escherichia coli urinary isolates and it is related to ESBL-production. A follow-up of fosfomycin resistance is required.

Critically Important Antimicrobial Resistance Trends in Salmonella Derby and Salmonella Typhimurium Isolated from the Pork Production Chain in Brazil: A 16-Year Period.

Knowledge about antimicrobial resistance in Salmonella is relevant due to its importance in foodborne diseases. We gathered data obtained over 16 years in the southern Brazilian swine production chain to evaluate the temporal evolution of halo for carbapenem, and the MIC for third-generation cephalosporins, fluoroquinolone, and polymyxin in 278 Salmonella Derby and Typhimurium isolates. All antimicrobial resistance assays were performed in accordance with EUCAST. To assess the diameter halo, we used a mixed linear model, and to assess the MIC, an accelerated failure time model for interval-censored data using an exponential distribution was used. The linear predictor of the models comprised fixed effects for matrix, serovar, and the interaction between year, serovar, and matrix. The observed halo diameter has decreased for ertapenem, regardless of serovars and matrices, and for the serovar Typhimurium it has decreased for three carbapenems. The MIC for ciprofloxacin and cefotaxime increased over 16 years for Typhimurium, and for Derby (food) it decreased. We did not find evidence that the MIC for colistin, ceftazidime, ciprofloxacin (Derby), or cefotaxime (food Typhimurium and animal Derby) has changed over time. This work gave an overview of antimicrobial resistance evolution from an epidemiological point of view and observed that using this approach can increase the sensitivity and timeliness of antimicrobial resistance surveillance.

Predominance and clonal spread of CTX-M-15 in cefotaxime-resistant Klebsiella pneumoniae in Korea and their association with plasmid-mediated quinolone resistance determinants

Introductionβ-lactams and fluoroquinolones are extensively used worldwide in the treatment of infections caused by Enterobacterales. In this study, we investigated the prevalence of extended-spectrum β-lactamases (ESBL), their correlation with plasmid-mediated quinolone resistance determinants (PMQR) and clonal distribution among the cefotaxime-resistant K. pneumoniae isolates. MethodsIn Korea, a total of 429 K. pneumoniae collected in 2015 were studied. Antimicrobial susceptibility test for cefotaxime, ciprofloxacin and levofloxacin was performed by broth microdilution method. By PCR and/or sequencing, mutations in gyrA and parC genes, PMQR genes and ESBL were identified. Multilocus-sequence-type (MLST) was determined for isolates harboring CTX-M-15. ResultsAmong the 149 K. pneumoniae showing cefotaxime MICs of >1 μg/ml, 142 (95.3%) isolates were ESBL-producers and CTX-M-15 was predominant (99 isolates). Among the 142 ESBL-producers, mutations in gyrA and parC were found in 112 (78.9%) and 93 isolates (65.5%), respectively. PMQR genes were detected in 141 isolates and the non-susceptibility rate to ciprofloxacin and levofloxacin was 95.1% (135/142) and 82.4% (117/142), respectively. The most frequently found PMQR combination was qnrB-aac(6′)-Ib-cr-oqxAB, (58/142, 40.8%). By MLST, four major STs/CC: ST48, ST392, ST307 and CC15 accounted for 67% of the CTX-M-15 producers and the prevalence of qnrB was significantly higher in these four major STs/CC than other groups (P = 0.004). Of note, we found the additive effect of PMQR genes; the more PMQR genes, the higher ciprofloxacin MICs. ConclusionsCTX-M-15 was predominant among the cefotaxime-resistant K. pneumoniae and co-harboring CTX-M-15 and PMQR genes, especially qnrB, seems to contribute the spread of high risk clones.

Cefotaxime resistance in invasive Haemophilus influenzae isolates in Germany 2016-19: prevalence, epidemiology and relevance of PBP3 substitutions.

Haemophilus influenzae can cause invasive infections, in which cefotaxime is among the first-line antibiotics for treatment. The prevalence of cefotaxime-resistant H. influenzae in Europe is reported to be on a low level. Nevertheless, systematic studies with a large set of invasive isolates are scarce. To provide prevalence data for cefotaxime resistance in invasive H. influenzae isolates in Germany 2016-19 and investigate the epidemiological relevance of PBP3 mutations known to elevate the cefotaxime MIC. Cefotaxime susceptibility of invasive H. influenzae isolates, collected in the national laboratory surveillance programme, was examined by gradient agar diffusion (GAD) testing. Cefotaxime resistance was determined according to EUCAST guidelines (resistance breakpoint MIC >0.125 mg/L). Therefore, the MIC for all resistant isolates was verified by broth microdilution method (BMD). WGS was performed to investigate the genetic relationship of cefotaxime-resistant isolates and to analyse alterations in the PBP3. An analysis of the geographic distribution of the resistant isolates was performed. From 2016 to 2019, the German National Reference Laboratory for Meningococci and H. influenzae received 2432 invasive H. influenzae isolates from blood and CSF. According to GAD results, 27 strains were resistant to cefotaxime. BMD confirmed the resistance in 22 of these isolates, which equals a prevalence of cefotaxime resistance of 0.90% in invasive H. influenzae in Germany. Among cefotaxime-resistant isolates cgMLST revealed three clusters. PBP3 analysis showed previously described mutations in our strains. In comparison with cefotaxime-susceptible strains, the alterations L389F and Y557H were significantly associated with cefotaxime resistance, but were not present in all resistant strains. Geographic analysis showed that the disease cases with cefotaxime-resistant H. influenzae were evenly spread throughout the population in Germany. Cefotaxime is still well suited for the treatment of invasive H. influenzae infections. Rarely occurring cefotaxime resistance is caused by sporadic mutations. The role of PBP3 mutations needs further investigation.

Pneumococcal colonization in healthy and hospitalized Sri Lankan children between 2 months to 2 years

Background: Colonization with Streptococcus pneumoniae is considered a pre-requisite for pneumococcal diseases. Evaluating the pneumococcal isolates that colonize children will give surrogate data on the circulating serotypes in the country. This study was conducted to identify the pneumococcal colonization rates among healthy children between 2 months and 2 years of age and children of the same age group who are hospitalized with respiratory symptoms identify the serotypes and sensitivities of the pneumococcal isolates Methods & Materials: This prospective descriptive study was conducted among two cohorts of children aged between 2 months and 2 years. (A) Healthy children attending immunization clinics and (B) children hospitalized with respiratory symptoms with or without fever. A nasopharyngeal swab (NPS) was collected from all participants. The NPS was cultured on sheep blood agar and pneumococcal isolates were identified with Optochin sensitivity and bile solubility testing. Microbroth dilution was used to determine sensitivity for cefotaxime and penicillin while disc diffusion was used to determine sensitivity for erythromycin and tetracycline. Multiplex PCRs prescribed by CDC and others were used to identify serotypes and serogroups. Results: Three hundred children each were recruited from the healthy cohort (HC) and inward cohort (IC). Pneumococcal colonization rate for the HC was 29.3% (n = 88) while it was 39.0% (n = 117) among the IC (p = 0.008, Chi-square test). The predominant serotypes identified were 19F (n = 45, 22.0%), 6B (n = 26, 12.7%), 23F (n = 21, 10.2%) and 6A (n = 19, 9.3%). Considering all 131 pneumococcal isolates together, penicillin MIC ranged from 0.015 to 4 while the cefotaxime MIC ranged from 0.015 to 16. MIC50 and MIC90 for penicillin and cefotaxime were 1 and 2 μg/mL. Non susceptibility rates for tetracycline and erythromycin were 70.7% and 75.1% respectively. Conclusion: Pneumococcal colonization rates found were considerably higher and the predominant serotypes identified 19F, 6B, 23F and 6A. Subtyping to serogroup 6 is ongoing. Acknowledgements: Research grant WI216479 through Pfizer for financial assistance.

Carbapenemase OXA-423: A Novel OXA-23 Variant in Acinetobacter baumannii.

PurposeA novel variant of OXA-23, named OXA-423, was identified in an Acinetobacter baumannii clinical isolate. The aim of this study was to analyse the resistance phenotype of OXA-423.MethodsThe A. baumannii strain WY-0713 was isolated from an intensive care unit patient. PCR was used to detect the blaOXA-23-like genes. Amplifying, cloning and sequencing were performed for the complete blaOXA-23-like. The novel blaOXA-423 and its ancestor blaOXA-23 were cloned into the expression vector pET-28b(+), and transformed into E. coli Rosetta (DE3) for antibiotic susceptibility testing. SDS-PAGE, modified Hodge test and CarbaNP test were used for detecting the expression of OXA-423 and OXA-23.ResultsPCR screening of A. baumannii WY-0713 was positive for blaOXA-23-like genes. Sequencing of the PCR product identified a novel blaOXA-23-like, named blaOXA-423 which encoding OXA-423. OXA-423 differed from OXA-23 by a crucial amino acid substitution (Val128Ala). The V128A substitution was located at the conserved active-site motifs SAV of OXA-23. Antibiotic susceptibility testing performed using isogenic E. coli showed that the MICs of E. coli Rosetta (pET-OXA-423) for penicillins and carbapenems were lower (reduced MICs 4-fold to 16-fold) than that of E. coli Rosetta (pET-OXA-23). The MICs of cefotaxime, ceftazidime and aztreonam for both transformants remained the same as the acceptor strain. Moreover, OXA-423 was slightly inhibited by sulbactam, clavulanic acid and tazobactam. SDS-PAGE analysis showed that OXA-423 and OXA-23 were conspicuously expressed. Modified Hodge test and CarbaNP test were positive demonstrated both of them were functional.ConclusionOXA-423, the first report of an amino acid substitution located at conserved active-site motifs of OXA-23, conferred lower MIC values of penicillins and carbapenems as compared with OXA-23, while without affecting the resistance profiles of expanded-spectrum cephalosporins and aztreonam.

Antibiotic resistance mechanisms in Acinetobacter spp. strains isolated from patients in a paediatric hospital in Mexico

ObjectivesThe aim of this study was to identify Acinetobacter spp. strains from paediatric patients, to determine their genetic relationship, to detect antibiotic resistance genes and to evaluate the role of efflux pumps in antibiotic resistance. MethodsA total of 54 non-duplicate, non-consecutive Acinetobacter spp. isolates were collected from paediatric patients. Their genetic relationship, antibiotic resistance profile, efflux pump activity, antibiotic resistance genes and plasmid profile were determined. ResultsThe isolates were identified as 24 Acinetobacter haemolyticus, 24 Acinetobacter calcoaceticus–baumannii (Acb) complex and 1 strain each of Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter indicus, Acinetobacter lwoffii, Acinetobacter ursingii and Acinetobacter venetianus. The 24 A. haemolyticus were considered genetically unrelated. One strain was resistant to carbapenems, two to cephalosporins, two to ciprofloxacin and sixteen to aminoglycosides. The antibiotic resistance genes blaOXA-214 (29%), blaOXA-215 (4%), blaOXA-264 (8%), blaOXA-265 (29%), blaNDM-1 (4%), aac(6ʹ)-Ig (38%) and the novel variants blaOXA-575 (13%), blaTEM-229 (75%), aac(6ʹ)-Iga (4%), aac(6ʹ)-Igb (13%) and aac(6ʹ)-Igc (42%) were detected. Among 24 Acb complex, 5 were multidrug-resistant, carbapenem-resistant strains carrying blaOXA-51 and blaOXA-23; they were genetically related and had the same plasmid profile. Other species were susceptible. In some strains of A. haemolyticus and Acb complex, the role of RND efflux pumps was evidenced by a decrease in the MICs for cefotaxime, amikacin and ciprofloxacin in the presence of an efflux pump inhibitor. ConclusionsThis study identified isolates of A. haemolyticus carrying new β-lactamase variants and shows for the first time the contribution of efflux pumps to antibiotic resistance in this species.

Genome analysis of enterobacteriaceae with non-wild type susceptibility to third-generation cephalosporins recovered from diseased dogs and cats in Europe

BackgroundExtended-spectrum-β-lactamases (ESBL) and plasmid-mediated cephalosporinases (pAmpC)-producing Enterobacteriaceae isolates are now reported worldwide in humans, animals, and in the environment. We identified the determinants of resistance to β-lactams and associated resistance genes as well as phylogenetic diversity of 53 ESBL- or pAmpC-producing Enterobacteriaceae isolated from dogs and cats in Europe. Materials/methodsOf a collection of 842 Enterobacteriaceae isolates that were recovered in 2013 and 2014 from 842 diseased and untreated dogs and cats, for 242 ampicillin or amoxicillin resistant isolates (MIC ≥ 16 mg/L), cefotaxime (CTX) and ceftazidime (CAZ) MICs were determined. Isolates with CTX and/or CAZ MIC ≥ 1 mg/L (n = 63) were selected, and their genomes were fully sequenced using Illumina Technology. Genomic data were explored to identify the resistance determinants, the plasmid incompatibility groups, and the sequence types (STs). Plasmid location of blaESBL and blaAmpC was evaluated for all isolates based on the co-localization of resistance and plasmid incompatibility group genes on the same contig. Phylogenetic trees were constructed using core-genome MLST. ResultsOf the 63 sequenced isolates, 53 isolates harbored a blaESBL or blaAmpC gene. Ten CTX and/or CAZ non-wild type isolates had neither blaESBL nor blaAmpC. Among the 63 isolates, 44 (69.8 %) were Escherichia coli, 11 (17.5 %) were Klebsiella pneumoniae, and 8 (12.7 %) were Proteus mirabilis. Fifty-one (80.9 %) isolates originated from dogs and 12 (19.1 %) from cats. Isolates were sampled from urinary tract (n = 36), skin and soft tissue (n = 22) and respiratory tract infections (n = 5). Thirty-two isolates (32/53, 60.4 %) carried blaESBL genes, including blaCTX-M-15 (n = 12), blaCTX-M-14 (n = 6), blaCTX-M-1 (n = 5), blaCTX-M-2 (n = 3), blaCTX-M-27 (n = 3), blaSHV-28 (n = 4), blaSHV-12 (n = 2), and blaVEB-6 (n = 1). Four isolates of K. pneumoniae had both blaCTX-M-15 and blaSHV-28. Twenty-one isolates (21/53, 39.6 %) carried genes encoding pAmpC, including blaCMY-2 (n = 19) and blaDHA-1 (n = 2). Thirteen E. coli isolates harbored both blaESBL or blaAmpC genes and plasmids of incompatibility groups IncIB (9/13), IncI1 (8/13), and IncFII (6/13). In addition to the reduced susceptibility to CTX and/or CAZ, reduced susceptibility or evidence of acquired resistance to at least one other relevant class of antibiotics was observed for all 63 isolates. E. coliisolates clustered in 23 STs, including B2 virulent clones from humans such as ST131 (n = 5), K. pneumoniae isolates mostly clustered in 3 STs: ST11 (n = 4), ST307 (n = 3), and ST16 (n = 2). Phylogenetic analysis identified the spread of E. coli ST131 blaCTX-M-27, and of K. pneumoniae ST307 harboring blaCTX-M-15 and blaSHV-28 or ST11 blaCTX-M-15. ConclusionsWe report here a 6.3 % prevalence of ESBL/pAmpC producing Enterobacteriaceae in diseased dogs and cats. This EU survey confirms that dogs and cats can be infected with epidemic multidrug resistant clones that may also spread in humans.

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC β-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization.

ObjectivesAmpC-β-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common β-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance.MethodsWe tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs.ResultsIn total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79–0.94) on the training set, 0.79 (95% CI=0.64–0.89) on validation set 1 and 0.85 (95% CI=0.71–0.94) on validation set 2.ConclusionsThis approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing.

In vitro activity of mecillinam and nitroxoline against Neisseria gonorrhoeae - re-purposing old antibiotics in the multi-drug resistance era.

In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l-1 and MICs of cefotaxime ranged from < 0.016 to 1 mg l-1 . Five isolates were penicillinase-producers. MICs of MCM ranged from 16 to > 128 mg l-1 whereas MICs of NIT ranged from 0.125 to 2 mg l-1 . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.

Characterization of ciprofloxacin-resistant and ESBL-producing Salmonella enteric serotype Derby in Eastern China

BackgroundFluoroquinolone resistance and ESBL-production are concurrently found in a limited number of Salmonella serotypes. The present study was aimed to characterize fluoroquinolone-resistant and ESBL-producing Salmonella enteric serotype Derby (S. Derby) isolates in terms of antimicrobial susceptibility, relevant genetic mechanisms, and PFGE.ResultsFrom 2013 to 2017 in Ningbo China, 52 S. Derby isolates were identified out of 826 non-typhoidal Salmonella isolates from patient feces, food, and environmental water samples. Three S. derby isolates were identified to be fluoroquinolone-resistant and ESBL-producing with cefotaxime MIC of 64 μg/mL and ciprofloxacin MIC of 4 μg/mL. The three isolates contained the same genetic structure of quinolone resistance, including a silent gyrA mutation S (TCC) 83S (TCT) and three PMQR genes qnrB, qnrS and aac(6′)-Ib-cr. As withβ-lactams resistance mechanisms, two isolates contained blaTEM, blaOXA, and blaCTX-M genes and one isolate contained blaOXA and blaCTX-M genes. Additionally, two isolates displayed more identical PFGE pattern than the third isolate, whereas three isolates showed the same plasmid profile of I1, W and P by PCR-based replicon typing. The conjugation experiment showed no dissemination of β-lactam resistance by direct contact among isolates; the transformation experiment failed to transfer plasmid conferring ampicillin resistance to E. coli DH5a.ConclusionThe present study demonstrates the emerging fluoroquinolone-resistant and ESBL-producing S. Derby in both humans and the environment. Seeing that S. Derby has become one of the most common Salmonella serotypes, this situation gives rise to a new major risk of food-borne diseases.

Cefotaxime-non-susceptibility of Haemophilus influenzae induced by additional amino acid substitutions of G555E and Y557H in altered penicillin-binding protein 3

Cefotaxime-non-susceptible Haemophilus influenzae has rarely been isolated from clinical specimens. Although several reports have shown that amino acid (AA) alteration in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, reduces activity of cefotaxime, precise mechanisms conferring the non-susceptibility have been unclear. We analyzed the ftsI gene of two clinically isolated cefotaxime-non-susceptible H. influenzae strains, 16-11 and 20-07 (minimum inhibitory concentrations [MICs]: 16 and 8 μg/mL, respectively), and found that their deduced AA sequences of PBP3 included two AA substitutions of G555E and Y557H in addition to previously described AA alterations. To clarify whether the two additional substitutions are requisite for cefotaxime non-susceptibility, we produced transformants of Rd KW20 (cefotaxime MIC: ≤0.06 μg/mL) with the ftsI gene of 16-11. Cefotaxime MICs against transformants M1 and M2, of which deduced PBP3s were altered with that of 16-11 entirely and partially (only the N-terminal side up to the AA position 519), were 8 and 0.25 μg/mL, respectively. We also produced M2-555/7 through site-directed mutagenesis inducing additional substitutions of G555E and Y557H into the PBP3 of M2, against which cefotaxime MIC was 8 μg/mL. These findings show that the additional substitutions of G555E and Y557H in PBP3 with previously described alterations cause cefotaxime non-susceptibility. An additional substitution of either G555E or Y557H alone in altered PBP3 reduced cefotaxime activity but the elevation of MICs were within the category of susceptibility. To our knowledge, this is the first study clarifying a genetic factor in the PBP3 causing cefotaxime non-susceptibility among H. influenzae strains.

Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil.

Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the blaCTX–M was the most prevalent (n = 12 positive isolates), followed by blaTEM (n = 5) and blaSHV (n = 4). blaCTX–M–15 (n = 5), blaCTX–M–14 (n = 1) and blaCTX–M–2 (n = 1) variants were detected in conserved genomic contexts: blaCTX–M–15 flanked by ISEcp1 and Orf477; blaCTX–M–14 flanked by ISEcp1 and IS903; and blaCTX–M–2 associated to an ISCR element. For 4 strains the transfer of blaCTX–M was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. blaCTX–M–15 as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., blaOXA–208 and blaADC–like). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains.

Potential effect of selective pressure with different β-lactam molecules on the emergence of reduced susceptibility to β-lactams in group B Streptococci.

In this study, the selective potential of group B Streptococcus isolates with reduced penicillin susceptibility (PRGBS) in a neonate-hypervirulent sequence type (ST)17 lineage was investigated by in vitro exposure to β-lactams. After 19 passages of stepwise penicillin exposure, PRGBS with a high penicillin minimum inhibitory concentration MIC (0.5 mg/L), greatly augmented ceftibuten MIC (>512 mg/L), and acquisition of G406D predicted to provide destabilizing effect (ΔΔG 0.099 kcal/mol) on PBP2X structure were identified. In early passages of stepwise cefotaxime exposure, PRGBS possessing G398E predicted to stabilize PBP2X (ΔΔG -0.038 kcal/mol) emerged with high MICs for cefotaxime (0.5 mg/L), ceftibuten (>512 mg/L) and penicillin (0.25 mg/L). Additionally, G398E + G329V + H438Y predicted to provide more stabilizing effect (ΔΔG -0.415 kcal/mol) were detected in mutants with higher MICs to cefotaxime (1 mg/L) and penicillin (0.5 mg/L). PRGBS mutants selected by penicillin and cefotaxime had a marked growth disadvantage compared with the parent strain. After two passages of stepwise ceftibuten exposure, the mutants exhibited increased MICs toward ceftibuten and acquisition of T555S predicted to provide stabilizing effect (ΔΔG -0.111 kcal/mol) in PBP 2X. In subsequent passages, gradual increases in ceftibuten MICs from 128 mg/L to 512 mg/L were found among selected mutants with accompanying stabilizing T555S+A354V (ΔΔG -0.257 kcal/mol) followed by stabilizing T555S + A354V + A536V (ΔΔG -0.322 kcal/mol), resulting in selection of a penicillin-susceptible group B Streptococcus lineage with reduced ceftibuten susceptibility (CTBr PSGBS). Notably, growth ability of CTBr PSGBS mutants was comparable to that of the parent strain. These findings may predict future failure of treatment for neonatal invasive infections caused by the neonate-hypervirulent PRGBS ST17 lineage.

Environmental superbugs: The case study of Pedobacter spp.

The environment is one of the main reservoirs of antibiotic resistance genes (ARGs) but multidrug resistant (MDR) environmental isolates are barely characterised. As suggested by the name, Pedobacter species have been predominantly isolated from soils, but are also recovered from water (including drinking water), chilled food, fish, compost, sludge, glaciers and other extreme environments. The susceptibility phenotype of Pedobacter lusitanus NL19 (isolated from a deactivated uranium mine), its closely related species and the genus type strain were investigated. All strains are MDR bacteria, resistant to β-lactams, colistin, aminoglycosides and ciprofloxacin. Therefore, Pedobacter spp. are likely intrinsically resistant to β-lactams (including ertapenem) and to other three classes of antibiotics. 6%–8% of their total protein-encoding genes encode a diverse collection of putative ARGs, including β-lactamases. These enzymes are highly abundant in all the other Pedobacter strains with sequenced genomes, especially class C, class B3 and class A. LUS-1 and PLN-1 were further characterised in E. coli. LUS-1 is a class A β-lactamase and it conferred an increase in the MIC of cefotaxime, albeit very low. PLN-1 is a class B3 β-lactamase with carbapenemase activity, conferring resistance to ertapenem and a 66x and 16x increase in the MIC of imipenem and meropenem, respectively. PLN-1 also hydrolyses ampicillin, 1st and 3rd generation cephalosporins, and at a lower extent cephamycins and 4th generation cephalosporins. Therefore, Pedobacter spp. encode a large and diverse arsenal of resistance mechanisms that make them environmental superbugs.

Antibiotic susceptibility and resistance profiles of Romanian Clostridioides difficile isolates

Abstract This study investigated the antibiotic susceptibility patterns and genetic resistance markers of 35 C. difficile strains isolated from patients with C. difficile infection. Vancomycin, metronidazole, tigecycline, teicoplanin, rifampicin, moxifloxacin, cefotaxime, tetracycline, erythromycin, clindamycin, chloramphenicol, linezolid and imipenem MICs were determined for toxigenic strains belonging to PCR ribotypes (PR) 012 (2), 014 (4), 017 (3), 018 (2), 027 (17), 046 (2), 087 (3) and 115 (2). Results showed vancomycin, metronidazole, tigecycline and teicoplanin to be active against all isolates. High resistance rates were noticed against cefotaxime (n = 35), clindamycin (n = 33), imipenem (n = 31), moxifloxacin (n = 25), erythromycin (n = 25) and rifampicin (n = 22). Linezolid-resistance was found in three isolates (PR 017/2, PR 012/1), showing complex resistance (7-9 antibiotics). PR 012, 017, 018, 027 and 046 isolates (n = 26) were resistant to 5-9 antibiotics. Twelve resistance profiles (2-9 antibiotics) were detected. Rifampicin-moxifloxacin-cefotaxime-erythromycin-clindamycin-imipenem-resistance was predominant, being expressed by 18 strains (PR 027/17, PR 018/1). PCR results suggested tetracycline-resistance to be induced by the gene tetM. Three tetM-positive isolates (PRs 012, 046), were also tndX-positive, suggesting the presence of a Tn5397-like element. Only two MLSB-resistant strains (PR 012) had the ermB gene and chloramphenicol-resistance determinant catD was not detected, leaving room for further investigating resistance mechanisms. Multidrug resistance could be attributed to most analysed strains, underlining, once more, the impact of wide-spectrum antimicrobial over prescription, still a tendency in our country, on transmission of antimicrobial resistance and emergence of epidemic C. difficile strains generating outbreaks.

Antimicrobial Susceptibility Patterns of Enteroaggregative E. coli, as the Most Common Diarrheagenic E. coli, Associated to Gastroenteritis Outbreaks in Iran

Background: Acute diarrhea, a leading cause of morbidity and mortality worldwide, still remains a major global health problem, especially among children in the developing countries. Diarrheagenic E. coli represent one of the most common etiological causes of diarrhea in children worldwide. Objectives: This study was conducted in order to determine the rate of Enteroaggregative E. coli (EAEC) among 50 E. coli isolates as well as its antimicrobial resistance patterns. Methods: A total of 50 Escherichia coli strains had been isolated among children under 5 years of age during 75 reported outbreaks in various provinces of Iran from October 2013 to May 2014. PCR was employed for the identification of different groups of diarrheagenic E. coli. Antimicrobial susceptibility testing was performed using disc diffusion methods. In addition, extended spectrum beta-lactamase (ESBL) production ability was checked by way of combination disc methods of CLSI. Minimum inhibitory concentration of cefotaxime, ceftriaxone, and ceftazidime in EAEC with the ability of ESBL production was determined using the micro-broth dilution method of CLSI. Results: Out of the 50 E. coli isolates, 17 were identified as Enteroaggregative E. coli (EAEC) in that they were positive for at least 1 of the 2 tested virulent genes: agg and aap. ESBL production ability was observed in 4/17 (23.5%) EAEC isolates. MIC of cefotaxime and ceftriaxone in ESBL positive EAEC varied between 8 - 32 µg/mL and 8 - 64 µg/mL, respectively. Resistance to ampicillin and nalidixic-acid (47.1%), trimethoprim/sulfamethoxazole (41.2%), and ciprofloxacin (11.8%) was observed among the EAEC isolates. No evidence of resistance to gentamycin and meropenem was detected. Conclusions: This research has revealed that the most common type of diarrheagenic E. coli among children, who were affected in the diarrheal outbreak in different cities of the country, is Enteroaggregative E. coli (34%). The rate of ESBL positive cases in EAEC isolates were 23.5 %.

Detection of blaCTX-M gene among Pseudomonas aeruginosa isolated from water samples in Baghdad

A total of 50 environmental Pseudomonas aeruginosa isolates were collected from sewage and tap water in Baghdad, Iraq. The MICs of Cefotaxime and Ceftazidime were determined by using agar dilution method, The MIC ranged from 2 to 256 µg/ml.The results of antibiotic sensitivity test showed that among sewage P. aeruginosa isolates, resistance was observed most often to Ticarcillin (92%), Penicillin G (84%), Ceftazidime (12%), (8%) for each of Cefotaxime and Ticarcillin. On the other hand, all tap water isolates were sensitive to Ofloxacin and Levofloxacin, Except (5%) of isolates were resistant to Cefotaxime (25%) to Ceftazidime and (95%) to Ticarcillin. All isolates were tested for Extended-Spectrum β-Lactamase (ESBL) production. Ten isolates (20%) were found to be ESBL producers. All environmental P. aeruginosa isolates were screened for the presence of the blaCTX-M genes by application PCR, Only (30%) of them were positive for this test.