The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

RNAIII is linked with the pentose phosphate pathway through the activation of RpiRc in Staphylococcus aureus.

Staphylococcus aureus RNAIII is a dual-function regulatory RNA that controls the expression of multiple virulence genes and especially the transition from adhesion to the production of exotoxins. However, its contribution to S. aureus central metabolism remains unclear. Using MS2-affinity purification coupled with RNA sequencing, we uncovered more than 50 novel RNAIII-mRNA interactions. Among them, we demonstrate that RNAIII is a major activator of the rpiRc gene, encoding a regulator of the pentose phosphate pathway (PPP). RNAIII binds the 5' UTR of rpiRc mRNA to favor ribosome loading, leading to an increased expression of RpiRc and, subsequently, of two PPP enzymes. Finally, we show that RNAIII and RpiRc are involved in S. aureus fitness in media supplemented with various carbohydrate sources related to PPP and glycolysis. Collectively, our data depict an unprecedented phenotype associated with the RNAIII regulon, especially the direct implication of RNAIII in central metabolic activity modulation. These findings show that the contribution of RNAIII in Staphylococcus aureus adaptation goes far beyond what was initially reported. IMPORTANCE: Staphylococcus aureus is a major human pathogen involved in acute and chronic infections. Highly recalcitrant to antibiotic treatment, persistent infections are mostly associated with the loss of RNAIII expression, a master RNA regulator responsible for the switch from colonization to infection. Here, we used the MS2 affinity purification coupled with RNA sequencing approach to identify novel mRNA targets of RNAIII and uncover novel functions. We demonstrate that RNAIII is an activator of the expression of genes involved in the pentose phosphate pathway and is implicated in the adjustment of bacterial fitness as a function of carbohydrate sources. Taken together, our results demonstrate an unprecedented role of RNAIII that goes beyond the knowledge gained so far and contributes to a better understanding of the role of RNAIII in bacterial adaptation expression and the coordination of a complex regulatory network.

Development of an ultrafast PCR to detect clinically relevant acquired vancomycin-resistance genes from cultured enterococci.

BACKGROUND: VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. METHODS: Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. RESULTS: All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). CONCLUSIONS: The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.

Impact of the phenotypic expression of temocillin resistance in Escherichia coli on temocillin efficacy in a murine peritonitis model.

BACKGROUND: Temocillin is a narrow spectrum ß-lactam active against MDR Enterobacterales. Mechanisms of acquired resistance to temocillin are poorly understood. We analysed resistance mechanisms in clinical isolates of Escherichia coli and evaluated their impact on temocillin efficacy in vitro and in a murine peritonitis model. METHODS: Two sets of isogenic clinical E. coli strains were studied: a susceptible isolate (MLTEM16S) and its resistant derivative, MLTEM16R (mutation in nmpC porin gene); and temocillin-resistant derivatives of E. coli CFT073: CFT-ΔnmpC (nmpC deletion), CFTbaeS-TP and CFTbaeS-AP (two different mutations in the baeS efflux-pump gene).Fitness cost, time-kill curves and phenotypic expression of resistance were determined. Temocillin efficacy was assessed in a murine peritonitis model. RESULTS: MICs of temocillin were 16 and 64 mg/L for MLTEM16S and MLTEM16R, respectively, and 8, 128, 256 and 256 mg/L for E. coli-CFT073, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP, respectively. No fitness cost of resistance was evidenced. All resistant strains showed heteroresistant profiles, except for CFTbaeS-AP, which displayed a homogeneous pattern. In vitro, temocillin was bactericidal against MLTEM16R, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP at 128, 256, 512 and 512 mg/L, respectively. In vivo, temocillin was as effective as cefotaxime against MLTEM16R, CFT-ΔnmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality). CONCLUSIONS: Heteroresistant NmpC porin alteration and active efflux modification do not influence temocillin efficacy despite high MIC values, unfavourable pharmacokinetic/pharmacodynamic conditions and the absence of fitness cost, whereas homogeneously expressed BaeS efflux pump alteration yielding similar MICs leads to temocillin inefficacy. MIC as sole predictor of temocillin efficacy should be used with caution.

Intracellular activity and in vivo efficacy in a mouse model of septic arthritis of the novel pseudopeptide Pep16 against Staphylococcus aureus clinical isolates.

Objectives: Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both in vitro and in vivo for the treatment of septic arthritis caused by Staphylococcus aureus. Methods: Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the in vivo efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg). Results: MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2 × MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (∼10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h. Conclusions: Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

Defining standard and high dosages for ß-lactam agents administered by intermittent, prolonged or continuous infusion: a PK/PD simulation study.

BACKGROUND: The new definitions of antimicrobial susceptibility categories proposed by EUCAST in 2020 require the definition of standard and high dosages of antibiotic. For injectable ß-lactams, standard and high dosages have been proposed for short-infusion regimens only. OBJECTIVES: To evaluate dosages for ß-lactams administered by prolonged infusion (PI) and continuous infusion (CI). METHODS: Monte Carlo simulations were performed for seven injectable ß-lactams: aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, piperacillin and temocillin. Various dosage regimens based on short infusion, PI or CI were simulated in virtual patients. Pharmacokinetic (PK) profiles and PTAs were obtained based on reference population PK models, as well as PK/pharmacodynamic targets and MIC breakpoints proposed by EUCAST. Alternative dosage regimens associated with PTA values similar to those of recommended dosages up to the breakpoints were considered acceptable. RESULTS: Adequate PTAs were confirmed for most EUCAST short-infusion dosage regimens. A total of 9 standard and 14 high dosages based on PI (3 to 4 h) or CI were identified as alternatives. For cefepime and aztreonam, only PI and CI regimens could achieve acceptable PTAs for infections caused by Pseudomonas spp.: 2 g q8h as PI of 4 h or 6 g/24 h CI for cefepime; 2 g q6h as PI of 3 h or 6 g/24 h CI for aztreonam. CONCLUSIONS: These alternative standard and high dosage regimens are expected to provide antibiotic exposure compatible with new EUCAST definitions of susceptibility categories and associated MIC breakpoints. However, further clinical evaluation is necessary.

Effectiveness of a multimodal intervention to improve blood culture collection in an adult emergency department.

We investigated the impact of a multimodal intervention to improve the compliance of BC collections as a composite outcome, taking into account both blood volume collected and absence of solitary BC. We performed a quasi-experimental study using a before-after design (5 months for pre- and post-intervention evaluation) in an adult emergency department at a tertiary care hospital that showed that a multimodal intervention was associated with a dramatic increase in the proportion of blood cultures that were collected as recommended per national guidelines, from 17.3% (328/1896) to 68.9% (744/1080), P < 0.0001. The implementation of such intervention in other settings could improve the diagnosis of bloodstream infections and reduce irrelevant costs.

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine.

Next-generation sequencing (NGS) has modernized the field of tuberculosis (TB) research by enabling high-throughput sequencing of the entire genome of Mycobacterium tuberculosis (MTB), which is the causative agent of TB. NGS has provided insights into the genetic diversity of MTB, which are crucial for understanding the evolution and transmission of the disease, and it has facilitated the identification of drug-resistant strains, enabling rapid and accurate tailoring of treatment. However, the high cost and the technical complexities of NGS currently limit its widespread use in clinical settings. International recommendations are thus necessary to facilitate the interpretation of polymorphisms, and an experimental approach is still necessary to correlate them to phenotypic data. This review aims to present a comparative, step-by-step, and up-to-date review of the techniques available for the implementation of this approach in routine laboratory workflow. Ongoing research on NGS for TB holds promise for improving our understanding of the disease and for developing more efficacious treatments.

Updated Review on Clinically-Relevant Properties of Delafloxacin.

The extensive use of fluoroquinolones has been consequently accompanied by the emergence of bacterial resistance, which triggers the necessity to discover new compounds. Delafloxacin is a brand-new anionic non-zwitterionic fluoroquinolone with some structural particularities that give it attractive proprieties: high activity under acidic conditions, greater in vitro activity against Gram-positive bacteria-even those showing resistance to currently-used fluoroquinolones-and nearly equivalent affinity for both type-II topoisomerases (i.e., DNA gyrase and topoisomerase IV). During phases II and III clinical trials, delafloxacin showed non-inferiority compared to standard-of-care therapy in the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia, which resulted in its approval in 2017 by the Food and Drug Administration for indications. Thanks to its overall good tolerance, its broad-spectrum in vitro activity, and its ease of use, it could represent a promising molecule for the treatment of bacterial infections.

Early detection of plasma d-lactate: Toward a new highly-specific biomarker of bacteraemia?

Background: Bloodstream infections are a leading cause of mortality. Their detection relies on blood cultures (BCs) but time to positivity is often between tens of hours and days. d-lactate is a metabolite widely produced by bacteria but very few in human. We aimed to evaluate d-lactate, d-lactate/l-lactate ratio and d-lactate/total lactate ratio in plasma as potential early biomarkers of bacteraemia on a strictly biological standpoint. Methods: A total of 228 plasma specimens were collected from patients who had confirmed bacteraemia (n = 131) and healthy outpatients (n = 97). Specific l-lactate and d-lactate analyses were performed using enzymatic assays and analytical performances of d-lactate, d-lactate/total lactate and d-lactate/l-lactate ratios for the diagnosis of bacteraemia were assessed. Results: A preliminary in vitro study confirmed that all strains of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus were able to produce d-lactate at significant levels. In patients, plasma d-lactate level was the most specific biomarker predicting a bacteraemia profile with a specificity and predictive positive value of 100% using a cut-off of 131 µmol.L-1. However, sensitivity and negative predictive value were rather low, estimated at 31% and 52%, respectively. d-lactate displayed an Area Under Receiver Operating Characteristic (AUROC) curve of 0.696 with a P value < 0.0001. There was no difference of d-lactate levels between BCs bottles positive for Gram-positive or Gram-negative bacteria (p = 0.55). Conclusion: d-lactate shows promise as a specific early biomarker of bacterial metabolism. The development of rapid automated assays could raise clinical applications for infectious diseases diagnosis including early bacteraemia prediction.

Recent Trends in Antimicrobial Resistance among Anaerobic Clinical Isolates.

Anaerobic bacteria are normal inhabitants of the human commensal microbiota and play an important role in various human infections. Tedious and time-consuming, antibiotic susceptibility testing is not routinely performed in all clinical microbiology laboratories, despite the increase in antibiotic resistance among clinically relevant anaerobes since the 1990s. ß-lactam and metronidazole are the key molecules in the management of anaerobic infections, to the detriment of clindamycin. ß-lactam resistance is usually mediated by the production of ß-lactamases. Metronidazole resistance remains uncommon, complex, and not fully elucidated, while metronidazole inactivation appears to be a key mechanism. The use of clindamycin, a broad-spectrum anti-anaerobic agent, is becoming problematic due to the increase in resistance rate in all anaerobic bacteria, mainly mediated by Erm-type rRNA methylases. Second-line anti-anaerobes are fluoroquinolones, tetracyclines, chloramphenicol, and linezolid. This review aims to describe the up-to-date evolution of antibiotic resistance, give an overview, and understand the main mechanisms of resistance in a wide range of anaerobes.

Simple purification and characterization of soluble and homogenous ABC-F translation factors from Enterococcus faecium.

The family of ATP-binding cassette F proteins (ABC-F) is mainly made up of cytosolic proteins involved in regulating protein synthesis, and they are often part of a mechanism that confers resistance to ribosome-targeting antibiotics. The existing literature has emphasized the difficulty of purifying these recombinant proteins because of their very low solubility and stability. Here, we describe a rapid and efficient three-step purification procedure that allows for the production of untagged ABC-F proteins from Enterococcus faecium in the heterologous host Escherichia coli. After four purified ABC-F proteins were produced using this protocol, their biological activities were validated by in vitro experiment. In conclusion, our study provides an invaluable tool for obtaining large amounts of untagged and soluble ABC-F proteins that can then be used for in vitro experiments.

Differential response to antibiotic therapy in staphylococcal infective endocarditis: contribution of an ex vivo model.

OBJECTIVES: Staphylococcal infective endocarditis (IE) remains a hard-to-treat infection with high mortality. Both the evaluation of new innovative therapies and research on alternative models mimicking human IE are therefore urgently needed to improve the prognosis of patients with diagnosed IE. Dalbavancin is a novel anti-staphylococcal lipoglycopeptide but there are limited data supporting its efficacy on biofilm infections. This antibiotic could be an alternative to current therapies for the medical treatment of IE but it needs to be further evaluated. METHODS: Here we developed an original ex vivo model of Staphylococcus aureus IE on human heart valves and assessed biofilm formation on them. After validating the model, the efficacy of two antistaphylococcal antibiotics, vancomycin and dalbavancin, was compared by measuring and visualizing their respective ability to inhibit and eradicate late-formed biofilm. RESULTS: Determination of the minimum biofilm inhibitory (MbIC) and eradicating (MbEC) concentrations in our ex vivo model identified dalbavancin as a promising drug with much lower MbIC and MBEC than vancomycin (respectively <0.01 versus 28 mg/L and 0.03 versus 32 mg/L). CONCLUSIONS: These data highlight a strong bactericidal effect of dalbavancin, particularly on an infected heart valve compared with vancomycin. Dalbavancin could be a realistic alternative treatment for the management of staphylococcal IE.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward ß-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

Synergistic Activity of Pep16, a Promising New Antibacterial Pseudopeptide against Multidrug-Resistant Organisms, in Combination with Colistin against Multidrug-Resistant Escherichia coli, In Vitro and in a Murine Peritonitis Model.

Colistin is a drug of last resort to treat extreme drug-resistant Enterobacterales, but is limited by dose-dependent toxicity and the emergence of resistance. A recently developed antimicrobial pseudopeptide, Pep16, which acts on the cell membrane, may be synergistic with colistin and limit the emergence of resistance. We investigated Pep16 activity against Escherichia coli with varying susceptibility to colistin, in vitro and in a murine peritonitis model. Two isogenic derivatives of E. coli CFT073 (susceptible and resistant to colistin) and 2 clinical isolates (susceptible (B119) and resistant to colistin (Af31)) were used. Pep16 activity, alone and in combination with colistin, was determined in vitro (checkerboard experiments, time-kill curves, and flow cytometry to investigate membrane permeability). Toxicity and pharmacokinetic analyses of subcutaneous Pep16 were performed in mice, followed by the investigation of 10 mg/kg Pep16 + 10 mg/kg colistin (mimicking human concentrations) in a murine peritonitis model. Pep16 alone was inactive (MICs = 32-64 mg/L; no bactericidal effect). A concentration-dependent bactericidal synergy of Pep16 with colistin was evidenced on all strains, confirmed by flow cytometry. In vivo, Pep16 alone was ineffective. When Pep16 and colistin were combined, a significant decrease in bacterial counts in the spleen was evidenced, and the combination prevented the emergence of colistin-resistant mutants, compared to colistin alone. Pep16 synergizes with colistin in vitro, and the combination is more effective than colistin alone in a murine peritonitis by reducing bacterial counts and the emergence of resistance. Pep16 may optimize colistin use, by decreasing the doses needed, while limiting the emergence of colistin-resistant mutants.

Evaluation of ceftolozane-tazobactam susceptibility on a French nationwide collection of Enterobacterales.

OBJECTIVES: Ceftolozane-tazobactam (C/T) proved its efficacy for the treatment of infections caused by non-carbapenemase producing Pseudomonas aeruginosa and Enterobacterales. Here, we aimed to provide susceptibility data on a large series of Enterobacterales since the revision of EUCAST categorization breakpoints in 2020. METHODS: First, C/T susceptibility was determined on characterized Enterobacterales resistant to third generation cephalosporins (3GCs) (extended spectrum ß-lactamase [ESBL] production or different levels of AmpC overexpression) (n = 213) and carbapenem-resistant Enterobacterales (CRE) (n = 259), including 170 carbapenemase producers (CPE). Then, 1632 consecutive clinical Enterobacterales responsible for infection were prospectively collected in 23 French hospitals. C/T susceptibility was determined by E-test® (biomérieux) and broth microdilution (BMD) (Sensititre™, Thermo Scientific) to perform method comparison. RESULTS: Within the collection isolates, 88% of 3GC resistant strains were susceptible to C/T, with important variation depending on the resistance mechanism: 93% vs. 13% susceptibility for CTX-M and SHV-ESBL producers, respectively. Only 20% of the CRE were susceptible to C/T. Among CPE, 80% of OXA-48-like producers were susceptible to C/T, whereas all metallo-ß-lactamase producers were resistant. The prospective study revealed that 95.6% of clinical isolates were susceptible to C/T. Method comparison performed on these 1632 clinical isolates demonstrated 99% of categorization agreement between MIC to C/T determined by E-test® in comparison with the BMD (reference) and only 74% of essential agreement. CONCLUSION: Overall, C/T showed good activity against wild-type Enterobacterales, AmpC producers, and ESBL-producing Escherichia coli but is less active against ESBL-producing Klebsiella pneumoniae, and CRE. E-test® led to an underestimation of the MICs in comparison to the BMD reference.

Cefotaxime resistance in bile samples is an independent predictor of deep infectious complications after pancreaticoduodenectomy in patients with endoprosthesis.

BACKGROUND: Bacteriobilia may increase the rate of deep infectious complications (DIC) after pancreaticoduodenectomy. To better adjust prophylactic and empirical antibacterial treatment, we aimed to characterize bacteriobilia in patients with preoperative endoprosthesis, and its association with postoperative DIC. METHODS: All patients who underwent pancreaticoduodenectomy in our center between 2010 and 2019 were included. The association between microbiological findings from bile samples, and postoperative DIC was analyzed, and we compared microbiology data between 2010-2014 and 2015-2019 periods. RESULTS: We enrolled 578 patients (median age 67 years [59-72], 58.7% males), of whom 220 (38.1%) had preoperative biliary endoprosthesis, with 197 (89.5%) positive preoperative bile samples pathogens were Enterobacterales, enterococci, and Candida albicans. The incidence of DIC was similar in patients with or without endoprosthesis (20.4% vs 17.8%, P = .352). Bacterial isolates collected during 2015-2019 were more resistant to cefotaxime than those recovered from 2010-2014 (45.5% vs 25.5%, P = .009). The only independent risk factor for DIC in patients with endoprosthesis was cefotaxime resistance in bile (hazard ratio 3.027 [1.115-8.216], P = .03). CONCLUSIONS: The incidence of DIC is high after pancreaticoduodenectomy, with or without endoprosthesis, despite routine postoperative treatment. Cefotaxime resistance, the only independent predictor of DIC in patients with endoprosthesis, has increased over time. Hence, cefotaxime may no longer be an appropriate empirical treatment.