Minimum Inhibitory Concentrations Of Cefotaxime Research Articles

Antimicrobial Use Survey and Detection of ESBL-Escherichia coli in Commercial and Medium-/Small-Scale Poultry Farms in Selected Districts of Zambia.

Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers' usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88-20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00-0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.

Efficacy of delafloxacin alone and in combination with cefotaxime against cefotaxime non-susceptible invasive isolates of Streptococcus pneumoniae.

We assessed the in vitro activity of delafloxacin and the synergy between cefotaxime and delafloxacin among cefotaxime non-susceptible invasive isolates of Streptococcus pneumoniae (CNSSP). A total of 30 CNSSP (cefotaxime MIC > 0.5 mg/L) were studied. Serotyping was performed by the Pneumotest-Latex and Quellung reaction. Minimum inhibitory concentrations (MICs) of delafloxacin, levofloxacin, penicillin, cefotaxime, erythromycin and vancomycin were determined by gradient diffusion strips (GDS). Synergistic activity of delafloxacin plus cefotaxime against clinical S. pneumoniae isolates was evaluated by the GDS cross method. Delafloxacin showed a higher pneumococcal activity than its comparator levofloxacin (MIC50, 0.004 versus 0.75 mg/L and MIC90, 0.047 versus >32 mg/L). Resistance to delafloxacin was identified in 7/30 (23.3%) isolates, belonging to serotypes 14 and 9V. Synergy between delafloxacin and cefotaxime was detected in 2 strains (serotypes 19A and 9V). Antagonism was not observed. Addition of delafloxacin increased the activity of cefotaxime in all isolates. Delafloxacin susceptibility was restored in 5/7 (71.4%) strains. CNSSP showed a susceptibility to delafloxacin of 76.7%. Synergistic interactions between delafloxacin and cefotaxime were observed in vitro among CNSSP by GDS cross method.

Contribution of genetic factors towards cefotaxime and ciprofloxacin resistance development among Extended spectrum beta-lactamase producing-Quinolone resistant pathogenic Enterobacteriaceae

β-lactams and quinolones are widely utilised to treat pathogenic Enterobacterial isolates worldwide. Due to improper use of these antibiotics, both ESBL producing and quinolone resistant (ESBL-QR) pathogenic bacteria have emerged. Nature of contribution of beta-lactamase (bla)/quinolone resistant (QR) genes, efflux pumps (AcrAB-TolC) over-expression and outer membrane proteins (OMPs) /porin loss/reduction and their combinations towards development of this phenotype were explored in this study. Kirby–Bauer disc diffusion method was used for phenotypic characterization of these bacteria and minimum inhibitory concentration of cefotaxime and ciprofloxacin was determined by broth micro dilution assay. Presence of bla, QR, gyrA/B genes was examined by PCR; acrB upregulation by real-time quantitative PCR and porin loss/reduction by SDS-PAGE. Based on antibiogram, phenotypic categorization of 715 non-duplicate clinical isolates was: ESBL+QR+ (n = 265), ESBL+QR- (n = 6), ESBL-QR+ (n = 346) and ESBL-QR-(n = 11). Increased OmpF/K35 and OmpC/K36 reduction, acrB up-regulation, prevalence of bla, QR genes and gyrA/B mutation was observed among the groups in following order: ESBL+QR+> ESBL-QR+> ESBL+QR-> ESBL-QR-. Presence of bla gene alone or combined porin loss and efflux pump upregulation or their combination contributed most for development of a highest level of cefotaxime resistance of ESBL+QR+ isolates. Similarly, combined presence of QR genes, porin loss/reduction, efflux pump upregulation and gyrA/B mutation contributed towards highest ciprofloxacin resistance development of these isolates.

Mortality associated with third-generation cephalosporin resistance in Enterobacterales bloodstream infections at eight sub-Saharan African hospitals (MBIRA): a prospective cohort study

Bacteria of the order Enterobacterales are common pathogens causing bloodstream infections in sub-Saharan Africa and are frequently resistant to third-generation cephalosporin antibiotics. Although third-generation cephalosporin resistance is believed to lead to adverse outcomes, this relationship is difficult to quantify and has rarely been studied in this region. We aimed to measure the effects associated with resistance to third-generation cephalosporins in hospitalised patients with Enterobacterales bloodstream infection in Africa. We conducted a prospective, matched, parallel cohort study at eight hospitals across sub-Saharan Africa. We recruited consecutive patients of all age groups with laboratory-confirmed Enterobacterales bloodstream infection and matched them to at least one patient without bloodstream infection on the basis of age group, hospital ward, and admission date. Date of infection onset (and enrolment) was defined as the day of blood sample collection for culturing. Patients infected with bacteria with a cefotaxime minimum inhibitory concentration of 1 mg/L or lower were included in the third-generation cephalosporin-susceptible (3GC-S) cohort, and the remainder were included in the third-generation cephalosporin-resistant (3GC-R) cohort. The primary outcomes were in-hospital death and death within 30 days of enrolment. We used adjusted multivariable regression models to first compare patients with bloodstream infection against matched patients within the 3GC-S and 3GC-R cohorts, then compared estimates between cohorts. Between Nov 1, 2020, and Jan 31, 2022, we recruited 878 patients with Enterobacterales bloodstream infection (221 [25·2%] to the 3GC-S cohort and 657 [74·8%] to the 3GC-R cohort) and 1634 matched patients (420 [25·7%] and 1214 [74·3%], respectively). 502 (57·2%) bloodstream infections occurred in neonates and infants (age 0-364 days). Klebsiella pneumoniae (393 [44·8%] infections) and Escherichia coli (224 [25·5%] infections) were the most common Enterobacterales species identified. The proportion of patients who died in hospital was higher in patients with bloodstream infection than in matched controls in the 3GC-S cohort (62 [28·1%] of 221 vs 22 [5·2%] of 420; cause-specific hazard ratio 6·79 [95% CI 4·06-11·37] from Cox model) and the 3GC-R cohort (244 [37·1%] of 657 vs 115 [9·5%] of 1214; 5·01 [3·96-6·32]). The ratio of these cause-specific hazard ratios showed no significant difference in risk of in-hospital death in the 3GC-R cohort versus the 3GC-S cohort (0·74 [0·42-1·30]). The ratio of relative risk of death within 30 days (0·82 [95% CI 0·53-1·27]) also indicated no difference between the cohorts. Patients with bloodstream infections with Enterobacterales bacteria either resistant or susceptible to third-generation cephalosporins had increased mortality compared with uninfected matched patients, with no differential effect related to third-generation cephalosporin-resistance status. However, this finding does not account for time to appropriate antibiotic treatment, which remains clinically important to optimise. Measures to prevent transmission of Enterobacterales could reduce bloodstream infection-associated mortality from both drug-resistant and drug-susceptible bacterial strains in Africa. Bill & Melinda Gates Foundation.

Penicillin-binding protein 1b encoded by mrcB gene mediates the enhancement of biofilm formation by subinhibitory concentrations of cefotaxime in monophasic Salmonella Typhimurium strain SH16SP46.

Development of cefotaxime-resistance and biofilm formation increase the difficulty to prevent and control the infection and contamination of Salmonella, one of the most important foodborne and zoonotic bacterial pathogen. Our previous study observed that 1/8 minimum inhibitory concentration (MIC) of cefotaxime induced the enhancement of biofilm formation and filamentous morphological change by a monophasic Salmonella Typhimurium strain SH16SP46. This study was designed to explore the role of three penicillin-binding proteins (PBPs) in mediating the induction effect of cefotaxime. Three deletion mutants of the genes mrcA, mrcB, and ftsI, encoding the proteins PBP1a, PBP1b, and PBP3, respectively, were constructed using the parental Salmonella strain SH16SP46. Gram staining and scanning electron microscopy showed that these mutants showed normal morphology comparable to the parental strain without cefotaxime treatment. However, under the stress of 1/8 MIC of cefotaxime, the strains WT, ΔmrcA, and ΔftsI, rather than ΔmrcB, exhibited filamentous morphological change. Moreover, cefotaxime treatment significantly enhanced biofilm formation by the strains WT, ΔmrcA, and ΔftsI, but not by the ΔmrcB strain. The complement of mrcB gene in the ΔmrcB strain recovered the enhanced biofilm formation and filamentous morphological change induced by cefotaxime. Our results suggest that PBP1b encoded by mrcB gene may be a binding target of cefotaxime for initiating the effect on Salmonella morphology and biofilm formation. The study will contribute to further understanding of the regulatory mechanism of cefotaxime on Salmonella biofilm formation.

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

Haemophilus influenzae is a causative agent of serious infections, especially among children. β-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, β-lactam resistance is due to the presence of β-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for β-lactams (BLNAR-β-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from β-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 β-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 β-lactam susceptibility in H. influenzae.

Variability in Adaptive Resistance of Salmonella Typhimurium to Sublethal Levels of Antibiotics.

This study was designed to evaluate the adaptive resistance of Salmonella Typhimurium under continuous sublethal selective pressure. Salmonella Typhimurium ATCC 19585 (STATCC) and S. Typhimurium CCARM 8009 (STCCARM) were sequentially cultured for 3 days at 37 °C in trypticase soy broth containing 1/2 × MICs of cefotaxime (CEF1/2), chloramphenicol (CHL1/2), gentamicin (GEN1/2), and polymyxin B (POL1/2). The STATCC and STCCARM exposed to CEF1/2, CHL1/2, GEN1/2, and POL1/2 were evaluated using antibiotic susceptibility, cross-resistance, and relative fitness. The susceptibilities of STATCC exposed to GEN1/2 and POL1/2 were increased by a 2-fold (gentamicin) and 8-fold (polymyxin B) increase in minimum inhibitory concentration (MIC) values, respectively. The MIC values of STCCARM exposed to CEF1/2, CHL1/2, GEN1/2, and POL1/2 were increased by 4-fold (cefotaxime), 2-fold (chloramphenicol), 2-fold (gentamicin), and 8-fold (polymyxin B). The highest heterogeneous fractions were observed for the STATCC exposed to CEF1/2 (38%) and POL1/2 (82%). The STCCARM exposed to GEN1/2 was cross-resistant to cefotaxime (p < 0.05), chloramphenicol (p < 0.01), and polymyxin B (p < 0.05). The highest relative fitness levels were 0.92 and 0.96, respectively, in STATCC exposed to CEF1/2 and STCCARM exposed to POL1/2. This study provides new insight into the fate of persistent cells and also guidance for antibiotic use.

In Vitro Antimicrobial Susceptibility Profiles of Gram-Positive Anaerobic Cocci Responsible for Human Invasive Infections.

The aim of this multicentre study was to determine the in vitro susceptibility to anti-anaerobic antibiotics of Gram-positive anaerobic cocci (GPAC) isolates responsible for invasive infections in humans. A total of 133 GPAC isolates were collected in nine French hospitals from 2016 to 2020. All strains were identified to the species level (MALDI-TOF mass spectrometry, 16S rRNA sequencing). Minimum inhibitory concentrations (MICs) of amoxicillin, piperacillin, cefotaxime, imipenem, clindamycin, vancomycin, linezolid, moxifloxacin, rifampicin, and metronidazole were determined by the reference agar dilution method. Main erm-like genes were detected by PCR. The 133 GPAC isolates were identified as follows: 10 Anaerococcus spp., 49 Finegoldia magna, 33 Parvimonas micra, 30 Peptoniphilus spp., and 11 Peptostreptococcus anaerobius. All isolates were susceptible to imipenem, vancomycin (except 3 P. micra), linezolid and metronidazole. All isolates were susceptible to amoxicillin and piperacillin, except for P. anaerobius (54% and 45% susceptibility only, respectively). MICs of cefotaxime widely varied while activity of rifampicin, and moxifloxacin was also variable. Concerning clindamycin, 31 were categorized as resistant (22 erm(A) subclass erm(TR), 7 erm(B), 1 both genes and 1 negative for tested erm genes) with MICs from 8 to >32 mg/L. Although GPACs are usually susceptible to drugs commonly used for the treatment of anaerobic infections, antimicrobial susceptibility should be evaluated in vitro.

Predicting Oral Beta-lactam susceptibilities against Streptococcus pneumoniae

BackgroundOral beta-lactam antimicrobials are not routinely tested against Streptococcus pneumoniae due to presumed susceptibility based upon penicillin minimum inhibitory concentration (MIC) testing. Currently, Clinical and Laboratory Standards Institute provides comments to use penicillin MIC ≤0.06 to predict oral cephalosporin susceptibility. However, no guidance is provided when cefotaxime MIC is known, leading to uncertainty with interpretation. The purpose of this study was to evaluate cefotaxime and penicillin MICs and their respective correlation to oral beta-lactam categorical susceptibility patterns.Methods249 S. pneumoniae isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF) and then tested by broth microdilution method to penicillin, cefotaxime, amoxicillin, cefdinir, cefpodoxime, and cefuroxime.ResultsUsing Clinical and Laboratory Standards Institute (CLSI) non-meningitis breakpoints for cefotaxime, 240/249 isolates were classified as susceptible. Of the cefotaxime susceptible isolates, 23% of the isolates are misrepresented as cefdinir susceptible. Amoxicillin correlated well with penicillin MIC breakpoints with only 1 discordant isolate out of 249.ConclusionThe correlation between amoxicillin and penicillin creates a very reliable predictor to determine categorical susceptibility. However oral cephalosporins were not well predicted by either penicillin or cefotaxime leading to the possible risk of treatment failures. Caution should be used when transitioning to oral cephalosporins in cefotaxime susceptible isolates, especially with higher cefotaxime MICs.

Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance.

Cefotaxime (CTX) is a third-generation cephalosporin (3GC) commonly used to treat infections caused by Escherichia coli . Two genetic mechanisms have been associated with 3GC resistance in E. coli . The first is the conjugative transfer of a plasmid harbouring antibiotic-resistance genes. The second is the introduction of mutations in the promoter region of the ampC β-lactamase gene that cause chromosome-encoded β-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to CTX resistance has not been reported. We recultured 172 cefoxitin-resistant E. coli isolates with known CTX minimum inhibitory concentrations and performed genome-wide analysis of homoplastic mutations associated with CTX resistance by comparing Illumina whole-genome sequencing data of all isolates to a PacBio sequenced reference chromosome. We mapped the mutations on the reference chromosome and determined their occurrence in the phylogeny, revealing extreme homoplasy at the −42 position of the ampC promoter. The 24 occurrences of a T at the −42 position rather than the wild-type C, resulted from 18 independent C>T mutations in five phylogroups. The −42 C>T mutation was only observed in E. coli lacking a plasmid-encoded ampC gene. The association of the −42 C>T mutation with CTX resistance was confirmed to be significant (false discovery rate <0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C>T mutation of the ampC promotor as significantly associated with CTX resistance and underlines the role of recurrent mutations in the spread of antibiotic resistance.

Pneumococcal colonization among healthy and hospitalized vaccine-naive Sri Lankan children

BackgroundStreptococcus pneumoniae continues to cause mortality and morbidity despite availability of effective vaccines. Pneumococcal colonization is considered a pre-requisite for disease. Identifying the serotypes circulating in a given locale is important for surveillance purposes as well as for assessing the need for vaccination.Aim of the present study was to identify nasopharyngeal pneumococcal colonization rates in healthy children and children with respiratory tract infections in central Sri Lanka. MethodA total of 450 nasopharyngeal swabs (NPS) of children aged between 2 months and 2 years were collected from two groups; healthy children and children hospitalized with respiratory symptoms. NPS samples were processed using conventional laboratory techniques to isolate S. pneumoniae. Antibiotic susceptibility patterns of pneumococcal isolates were identified using CLSI disc diffusion method and minimum inhibitory concentration (MIC) was determined by micro-broth dilution method. ResultsPneumococcal colonization rate among healthy children was 31.8% (143/450) it was 39.8% (179/450) in children hospitalized with respiratory symptoms. MIC for penicillin and cefotaxime ranged between 0.015 to 4 µg/ml and <0.015 to 16 µg/ml respectively. All isolates were susceptible to levofloxacin, vancomycin, linezolid and rifampicin. Erythromycin and tetracycline non-susceptibility rates were >50% in both groups. The predominant serotypes identified were 19F (n = 66, 20.5%), 6B (n = 43, 13.4%), 6A (n = 30, 9.3%), 23F (n = 28, 8.7%) and 14 (n = 20, 6.2%). Among healthy children, presence of school going children at home and the number of household members were significantly associated with pneumococcal colonization while in hospitalized children, pneumococcal colonization was significantly associated with presence of school going children at home. ConclusionPneumococcal colonization rates were considerably higher in both study cohorts and the commonest serotypes were 19F, 6B, 6A, 23F and 14. Antibiotic resistance rates were also relatively higher among the pneumococcal isolates.

Multidrug-Resistant Streptococcus agalactiae Strains Found in Human and Fish with High Penicillin and Cefotaxime Non-Susceptibilities.

Penicillin non-susceptible Streptococcus agalactiae (PEN-NS GBS) has been increasingly reported, with multidrug-resistant (MDR) GBS documented in Japan. Here we identified two PEN-NS GBS strains during our surveillance studies: one from a patient’s wound and the other from a tilapia. The patient’s GBS (H21) and fish GBS (F49) were serotyped and tested for antibiotic susceptibility. Whole-genome sequencing was performed to find the sequence type, antimicrobial resistance genes, and mutations in penicillin-binding proteins (PBPs) and fluoroquinolone (FQ) resistance genes. H21 and F49 belonged to ST651, serotype Ib, and ST7, serotype Ia, respectively. H21 showed PEN and cefotaxime minimum inhibitory concentrations (MICs) of 2.0 mg/L. F49 showed PEN MIC 0.5 mg/L. H21 was MDR with ermB, lnuB, tetS, ant6-Ia, sat4a, and aph3-III antimicrobial resistance genes observed. Alignment of PBPs showed the combination of PBP1B (A95D) and 2B mutations (V80A, S147A, S160A) in H21 and a novel mutation in F49 at N192S in PBP2B. Alignment of FQ-resistant determinants revealed mutation sites on gyrA, gyrB, and parC and E in H21. To our knowledge, this is the first report of GBS isolates with such high penicillin and cefotaxime MICs. This raises the concern of emergence of MDR and PEN-NS GBS in and beyond healthcare facilities.

Streptococcus pyogenes pbp2x Mutation Confers Reduced Susceptibility to β-Lactam Antibiotics

Two near-identical clinical Streptococcus pyogenes isolates of emm subtype emm43.4 with a pbp2x missense mutation (T553K) were detected. Minimum inhibitory concentrations (MICs) for ampicillin and amoxicillin were 8-fold higher, and the MIC for cefotaxime was 3-fold higher than for near-isogenic control isolates, consistent with a first step in developing β-lactam resistance.

Cinnamaldehyde: a compound with antimicrobial and synergistic activity against ESBL-producing quinolone-resistant pathogenic Enterobacteriaceae.

Usage of cephalosporin and quinolone antibiotics has aggravated the development of extended-spectrum beta-lactamase (ESBL)-producing quinolone-resistant (QR) pathogenic Enterobacteriaceae. The present study aims to determine antimicrobial activity of cinnamaldehyde alone or in combination with cefotaxime/ciprofloxacin to reverse the drug resistance and evaluations of efficacy, and possible molecular mechanism of action of the combination was also evaluated using in vitro assays. Broth microdilution assay was used to determine minimum inhibitory concentrations (MICs) of cinnamaldehyde and antibiotics against ESBL-QR Enterobacteriaceae. Synergistic effect and dynamic interaction with antibiotics were further examined by checkerboard assay, isobologram analysis, and time-kill assay, respectively. Cellular morphology of bacteria was viewed with scanning electron microscopy (SEM). Effects of cinnamaldehyde and its combination on the expression of gene encoding-porins (ompC, ompF, ompK35, and ompK36), efflux pump genes (acrB-E. coli, acrB-K. pneumoniae), and antibiotic-resistant genes (blaTEM, blaSHV, blaCTXM, and QnrB) were evaluated using real-time quantitative PCR (RT-qPCR). Majority of the E. coli (32.1%) and K. pneumoniae (24.2%) isolates demonstrated MIC of cinnamaldehyde at 7.34μg/mL and 0.91g/mL, respectively. Synergism between cinnamaldehyde and cefotaxime was noted among 75% E. coli and 60.6% K. pneumoniae. Similarly, synergism with ciprofloxacin was observed among 39.6% and 42.4% of the bacteria, respectively. Thus, cinnamaldehyde reduced MIC of cefotaxime and ciprofloxacin 2-1024-fold with bactericidal and/synergistic effect after 24h. Cinnamaldehyde and its combination altered gene expression by ~ 1.6 to ~ 400-fold. Distorted bacterial cell structures were visible after treatment with cinnamaldehyde and/or with cefotaxime/ciprofloxacin. The results indicated the potential efficacy and mode of action of cinnamaldehyde alone and in combination with antibiotics against pathogenic ESBL-QR bacteria.

Short communication: N-Acetylcysteine–mediated augmentation of β-lactam antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from bovine mastitis cases

The present study investigated the effects of N-acetylcysteine (NAC) on β-lactam antibacterial activity against 20 methicillin-resistant Staphylococcus aureus (MRSA) isolates from bovine mastitis. Minimum inhibitory concentrations (MIC) were determined by the E-test method. The presence of 10 mM NAC reduced the MIC of penicillin, ampicillin, oxacillin, cefoxitin, ceftazidime, and cefotaxime to MRSA. Importantly, the MIC of cefoxitin in MRSA in the presence of NAC was lower than the susceptible breakpoint of cefoxitin. The results provide a new way to use current β-lactam antibiotics combined with NAC against MRSA.

An alarmingly high nasal carriage rate of Streptococcus pneumoniae serotype 19F non-susceptible to multiple beta-lactam antimicrobials among Vietnamese children

BackgroundUnderstanding the relationship between serotype epidemiology and antimicrobial susceptibility of Streptococcus pneumoniae is essential for the effective introduction of pneumococcal conjugate vaccines (PCVs) and control of antimicrobial-resistant pneumococci.MethodsWe conducted a community-based study in Nha Trang, central Vietnam, to clarify the serotype distribution and pattern of S. pneumoniae antimicrobial susceptibility in children under 5 years of age and to identify risk factors for carrying antimicrobial-resistant strains. Nasopharyngeal swabs collected from children with acute respiratory infections (ARIs) hospitalized between April 7, 2008, and March 30, 2009, and from healthy children randomly selected in July 2008 were subjected to bacterial culture. Minimum inhibitory concentrations (MICs) against S. pneumoniae were determined, and multiplex-polymerase chain reaction (PCR) serotyping assays were performed. Logistic regression was applied to identify risk factors.ResultsWe collected 883 samples from 331 healthy children and 552 ARI cases; S. pneumoniae was isolated from 95 (28.7%) healthy children and 202 (36.6%) ARI cases. Age and daycare attendance were significantly associated with pneumococcal carriage. In total, 18.0, 25.8 and 75.6% of the isolates had high MICs for penicillin (≥4 μg/ml), cefotaxime (≥2 μg/ml) and meropenem (≥0.5 μg/ml), respectively. The presence of pneumococci non-susceptible to multiple beta-lactams was significantly associated with serotype 19F (Odds Ratio: 4.23) and daycare attendance (Odds Ratio: 2.56) but not ARIs, age or prior antimicrobial use. The majority of isolates non-susceptible to multiple beta-lactams (90%) were PCV13 vaccine serotypes.ConclusionsS. pneumoniae serotype 19F isolates non-susceptible to multiple beta-lactams are widely prevalent among Vietnamese children. Vaccine introduction is expected to significantly increase drug susceptibility.

Evaluation of synergism between Cefotaxime and Allium sativum against Extended-spectrum beta-lactamase and Ambler class C co-producers

In vitro study on the efficacy of zinc oxide and titanium dioxide nanoparticles against metallo beta-lactamase and biofilm producing Pseudomonas aeruginosaMary Grace Vincent, Neetu Peedikayil John, Narayanan PM, Vani C and Sevanan Murugan

Molecular Characteristics of "BlaB-Like" Chromosomal Inducible Cephalosporinase of Yersinia enterocolitica Biotype 1A Strains.

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal β-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A produced a "BlaB-like" enzyme. The objective of this work was to study the molecular characteristics of "Bla-B like" β-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type β-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the "BlaB-like" enzymes were modeled, and docked in silico with cefotaxime to understand how specific substitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key catalytic residues were present in variants of "Bla-B-like" enzymes, discerned in biotype 1A strains, but at different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of "BlaB-like" cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.

Role of non-active site residues in maintaining New Delhi metallo-β-lactamase-1(NDM-1) function: an approach of site directed mutagenesis and docking.

New Delhi metallo-β-lactamase-1(NDM-1) has been known to hydrolyze nearly all β-lactam antibiotics, leading to multi drug resistant state. Hence, it is important to search its structure-function relation to control infections caused by such resistant bacterial strains. Mutagenesis is one of the approaches to explore it. No study has been performed to explore the role of non-active-site residues in the enzyme activity. This study includes mutations of three non-active site residues to comprehend its structure and function simultaneously. Three non-active-site laboratory mutants of NDM-1 were generated by site-directed mutagenesis. The MICs of cefotaxime, cefoxitin,imipenem and meropenem were reduced up to 4 fold for these mutants as compared to wild-type. The hydrolytic activity of mutants was also found to be reduced. Mutants showed a significant change in its secondary structure compared to wild type, as determined by CD spectrophotometer. Catalytic property and stability of these mutants were found to be reduced. Hence it revealed an imperative role of non-active-site residues in the enzymatic activity of NDM-1.

Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase.

Members of Enterobacteriaceae cause antibiotic-resistant infections worldwide. One such marker, CTX-M-15, expressed by Enterobacteriaceae produces β-lactamases, which hydrolyze the cephalosporin group of antibiotics, such as cefotaxime, used in the treatment of both Gram-positive and negative bacterial infections. Amino acid residues present in close proximity of the active site might also play a major role in the structure and function of CTX-M-15, hence the objective of this study was to investigate the significance of two amino acid residues, Asn-247 and Arg-64, present near to the active site in the hydrolysis of cefotaxime. blaCTX-M-15, cloned from the E. cloacae strain, and using Polymerase Chain Reaction (PCR)-based site-directed mutagenesis, Asn247Val and Arg64Leu mutations were introduced. The minimum inhibitory concentrations of cefotaxime for the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were reduced by 512 and 128 fold, respectively. Proteins/enzymes of wild-type CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were expressed and purified. Kinetic studies showed that the catalytic efficiencies of the N247V mutant and R64L mutant enzymes in the hydrolysis of cefotaxime were reduced by 89.66% and 71.11%, respectively. Circular dichroism spectroscopic studies showed considerable changes in the α-helical content of the mutant enzymes. A fluorescence study showed that N247V mutant-cefotaxime and R64L mutant-cefotaxime underwent complex formation with strong interactions. The study provides an understanding of the crucial role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the hydrolytic mechanism of CTX-M-15 type β-lactamases. Hence, Asn-247 and Arg-64 can be used as potential target sites for the design of inhibitory molecules against CTX-M-15-producing bacterial strains.