In vitro efficacy of combinations of antibiotics used in clinical practice on clinical isolates of Helicobacter pylori.

BACKGROUND: The main antibiotics used against Helicobacter pylori have been chosen empirically over time, with few preclinical studies to provide support. The rise in resistance to some of these antibiotics is prompting a reassessment of their use. This work aimed to evaluate the in vitro efficacy of 2 × 2 combinations of the most widely used antibiotics against H. pylori. MATERIALS AND METHODS: J99 reference strains and 19 clinical isolates of H. pylori with various antibiotic resistance phenotypes were used. Minimum inhibitory concentrations were carried out using the microdilution method in 96-well plates. The activity of 15 possible combinations of two antibiotics including amoxicillin, clarithromycin (CLA), levofloxacin, rifampicin, tetracycline, and metronidazole was determined for all strains by the checkerboard method. A mean fractional inhibitory concentration index (FICmean) was calculated for each combination and strain and the type of pharmacodynamic interaction was considered as synergic if FICmean ≤ 0.5, additive if 0.5 < FICmean ≤ 1, indifferent if 1 < FICmean < 4 or antagonistic if FICmean ≥ 4. RESULTS: Most of the 285 pharmacodynamic interactions tested with clinical strains were close to additivity (average FICmean = 0.89 [0.38-1.28]). No interaction was found to be antagonistic. When two antibiotics to which a strain was resistant were combined, the concentrations required to inhibit bacterial growth were higher than their respective breakpoints. CONCLUSION: The present results have shown that in vitro, the different antibiotics used in therapeutics have additive effects. The addition of the effects of two antibiotics to which a strain was resistant was not sufficient to inhibit bacterial growth. In probabilistic treatment, the choice of antibiotics to combine should therefore be based on the local epidemiology of resistance, and on susceptibility testing in the case of CLA therapy, so that at least one antibiotic to which the strain is susceptible is used.

Bacteremia due to Lachnoanaerobaculum umeaense in a patient with acute myeloid leukemia during chemotherapy: A case report, and a review of the literature.

The present case reports a bacteremia due to Lachnoanaerobaculum umeaense (a Gram-positive, filamentous, rod-shaped, anaerobic, spore-forming bacillus present in the human oral microbiota) in a patient treated for acute myeloid leukemia. After failed identification by MALDI-TOF, identification was done by sequencing of 16s rRNA. The patient was successfully treated with Amoxicillin-clavulanic acid and ciprofloxacin for seven days. Comparison of V1-V3 regions of the bacterial 16S rRNA gene gene with published sequences failed to classify the strain as pathogenic or non-pathogenic based on this phylogenetic classification alone. Although Lachnoanaerobaculum gingivalis are known to be associated with bacteremia in patients with acute myeloid leukemia, this clinical case of infection by L. umeaense argues for further studies that will lead to more efficient classification of the infection by these microorganisms.

The First Lethal Infection by Oligella ureolytica: A Case Report and Review of the Literature.

Oligella ureolytica is a Gram-negative bacillus, a member of the Alcaligenaceae family, that had never previously been reported as lethal. Herein, a case of fatal infection caused by Oligella ureolytica in an elderly woman with suspected bladder cancer is reported. The species identification was confirmed through Sanger sequencing of the bacterial 16S rRNA sequence and compared to published sequences for phylogenetic analysis. Initial antibiotic therapy with ceftriaxone and oxacillin was initiated but had to be switched due to resistance. Cefepime in combination with metronidazole was administered, unfortunately failing to prevent the patient's death. Further studies are needed to explore additional factors influencing clinical outcomes in Oligella ureolytica infections.

French national epidemiology of bacterial superinfections in ventilator-associated pneumonia in patients infected with COVID-19: the COVAP study.

BACKGROUND: Description and comparison of bacterial characteristics of ventilator-associated pneumonia (VAP) between critically ill intensive care unit (ICU) patients with COVID-19-positive, COVID + ; and non-COVID-19, COVID-. METHODS: Retrospective, observational, multicenter study that focused on French patients during the first wave of the pandemic (March-April 2020). RESULTS: 935 patients with identification of at least one bacteriologically proven VAP were included (including 802 COVID +). Among Gram-positive bacteria, S. aureus accounted for more than two-thirds of the bacteria involved, followed by Streptococcaceae and enterococci without difference between clinical groups regarding antibiotic resistance. Among Gram-negative bacteria, Klebsiella spp. was the most frequently observed bacterial genus in both groups, with K. oxytoca overrepresented in the COVID- group (14.3% vs. 5.3%; p < 0.05). Cotrimoxazole-resistant bacteria were over-observed in the COVID + group (18.5% vs. 6.1%; p <0.05), and after stratification for K. pneumoniae (39.6% vs. 0%; p <0.05). In contrast, overrepresentation of aminoglycoside-resistant strains was observed in the COVID- group (20% vs. 13.9%; p < 0.01). Pseudomonas sp. was more frequently isolated from COVID + VAPs (23.9% vs. 16.7%; p <0.01) but in COVID- showed more carbapenem resistance (11.1% vs. 0.8%; p <0.05) and greater resistance to at least two aminoglycosides (11.8% vs. 1.4%; p < 0.05) and to quinolones (53.6% vs. 7.0%; p <0.05). These patients were more frequently infected with multidrug-resistant bacteria than COVID + (40.1% vs. 13.8%; p < 0.01). CONCLUSIONS: The present study demonstrated that the bacterial epidemiology and antibiotic resistance of VAP in COVID + is different from that of COVID- patients. These features call for further study to tailor antibiotic therapies in VAP patients.

First Bacteremia Due to Corynebacterium gottingense in an Immunocompromised Child: A Case Report, 16S rDNA-Based Phylogenetic Analyses and Review of the Literature.

Corynebacterium gottingense is a Gram-positive bacillus that has not been reported as pathogenic in pediatric patients. Herein, a case of catheter-associated bloodstream infection by C. gottingense in a 13-year-old immunocompromised child with febrile neutropenia induced for osteosarcoma is reported. The species was identified by Sanger sequencing of the 16s rRNA sequence of the bacterial strain and was compared phylogenetically with published sequences. As suggested in the literature, the presented strain was multi-susceptible, particularly to amoxicillin. The patient was treated with piperacillin/tazobactam for seven days in the context of a urinary co-infection, resulting in resolution of fever within 48 h and then relaunched with oral amoxicillin for 3 days (for a total of 10 days of antibiotic therapy). Phylogenetic analyses based on 16S rDNA demonstrated the complexity of the genus Corynebacterium spp. but failed to demonstrate a direct benefit in predicting clinical outcome based on this single information.

How Did We End Up Using a Quadruple Guided Therapy Combining At Least Two Antibiotics with a PPI to Eradicate Helicobacter pylori?

Since the discovery of Helicobacter pylori, and even if the species is frequently susceptible to many antibiotics in vitro, only six of them (amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and rifabutin) and bismuth salts could be considered as effective in vivo to eliminate H pylori and have been used in recommended eradication treatments [...].

Phenol-soluble modulins α are major virulence factors of Staphylococcus aureus secretome promoting inflammatory response in human epidermis.

Staphylococcus aureus is a skin commensal microorganism commonly colonizing healthy humans. Nevertheless, S. aureus can also be responsible for cutaneous infections and contribute to flare-up of inflammatory skin diseases such as atopic dermatitis (AD), which is characterized by dysbiosis of the skin microbiota with S. aureus as the predominant species. However, the role of major virulence factors of this pathogen such as phenol-soluble modulin (PSM) toxins in epidermal inflammation remains poorly understood. Stimulation of primary human keratinocytes with sublytic concentrations of synthetic and purified PSM α3 resulted in upregulation of a large panel of pro-inflammatory chemokine and cytokine gene expression, including CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CCL20, IL-1α, IL-1ß, IL-6, IL-36γ and TNF-α, while inducing the release of CXCL8, CCL20, TNF-α and IL-6. In addition, using S. aureus culture supernatant from mutants deleted from genes encoding either α-type PSMs or all PSM production, PSMs were shown to be the main factors of S. aureus secretome responsible for pro-inflammatory mediator induction in human keratinocytes. On the other hand, α-type PSM-containing supernatant triggered an intense induction of pro-inflammatory mediator expression and secretion during both topical and basal layer stimulation of an ex vivo model of human skin explants, a physiologically relevant model of pluristratified epidermis. Taken together, the results of this study show that PSMs and more specifically α-type PSMs are major virulence factors of S. aureus inducing a potent inflammatory response during infection of the human epidermis and could thereby contribute to AD flare-up through exacerbation of skin inflammation.

K1 Antigen Is Associated with Different AST Profile in Escherichia coli: A One-Month-Long Pilot Study.

Escherichia coli is responsible for diseases of varying severity. The "K" antigen designates the capsular polysaccharides on the bacterial surface, which are mostly similar to those of highly pathogenic bacteria. The K1 antigen is often found in pathogenic E. coli. Aim: While the published studies on the AST profile of K1-positive E. coli have focused on pregnant women or newborns, this study aimed to characterize the AST profile of K1-positive E. coli independently of the clinical sample of isolation. Over a 4-week-long period, all patients hospitalized/consulting at the Poitiers University Hospital presenting a determined AST on E. coli were prospectively included to define their K1-status (Pastorex Meningitis) and to collect the clinical (age/sex) or biological metadata (AST/MIC). Among the 296 included samples, no differential representation was observed between K1 results regarding sample nature. K1-negative results were associated with multiple antibiotic-resistance (12.3% vs. 33.0%; p < 0.01). AST phenotypes differed between these groups, with a higher proportion of K1-negativity among resistant strains, especially on ß-lactams (ureidopenicillin, 25.8% vs. 14.9%; and ampicillin/inhibitor, 50.0% vs. 26.8%; p < 0.05) or quinolone (19.8% vs. 7.0%) and sulfamethoxazole-trimethoprim (30.2% vs. 12.3%) (p < 0.01). This study analyzed E. coli ASTs in clinical samples of all types, regarding their K1-antigen status.

Invasive pneumococcal disease incidence in children and adults in France during the pneumococcal conjugate vaccine era: an interrupted time-series analysis of data from a 17-year national prospective surveillance study.

BACKGROUND: The long-term benefits of pneumococcal conjugate vaccines (PCVs) remain unknown because of serotype replacement. We aimed to estimate the effect of PCV implementation on invasive pneumococcal disease incidence in France. METHODS: We did a quasi-experimental interrupted time-series analysis using data from a French national prospective surveillance system. We included all invasive pneumococcal disease cases in children and adults from more than 250 participating hospitals between Jan 1, 2001, and Dec 31, 2017. The primary outcome was incidence of invasive pneumococcal disease (meningitis and non-meningitis) over time, analysed by segmented regression with autoregressive error. Isolates were serotyped by latex agglutination with antiserum samples. FINDINGS: We included 75 903 patients with invasive pneumococcal disease, including 4302 (5·7%) children younger than 2 years and 37 534 (49·4%) adults aged 65 years or older. Before PCV7 implementation, the estimated monthly incidence of invasive pneumococcal disease was 0·78 cases per 100 000 inhabitants, which did not change significantly up to May, 2010. PCV13 implementation in 2010 was followed by a significant decrease in the incidence of invasive pneumococcal disease (-1·5% per month, 95% CI -2·2 to -0·8), reaching an estimated monthly incidence of 0·52 cases per 100 000 inhabitants in December, 2014. From January, 2015, the incidence rebounded (1·8% per month, 95% CI 1·0 to 2·6), reaching an estimated monthly incidence of 0·73 cases per 100 000 inhabitants in December, 2017. The estimated monthly incidence increased from 0·93 cases per 100 000 in December, 2014, to 1·73 cases per 100 000 in December, 2017, for children younger than 2 years, and from 1·54 cases per 100 000 in December, 2014, to 2·08 cases per 100 000 in December, 2017, for adults aged 65 years or older. The main non-PCV13 serotypes involved in the increase were 24F in young children and 12F, 22F, 9N, and 8 in adults aged 65 years or older. INTERPRETATION: PCV13 implementation led to a major reduction in the incidence of invasive pneumococcal disease. However, a rebound in cases among children and adults since 2015, driven by several emerging non-PCV13 serotypes, jeopardises the long-term PCV benefits. These findings, if confirmed in the coming years, should be considered in the development of next-generation PCVs and might guide policy makers in the selection of future pneumococcal vaccines. FUNDING: Foundation for Medical Research; Pfizer, BioMérieux, Sanofi for the Regional Observatory of Pneumococci.

Where to Biopsy to Detect Helicobacter pylori and How Many Biopsies Are Needed to Detect Antibiotic Resistance in a Human Stomach.

This study aims to determine the gastric distribution, density, and diversity of Helicobacter pylori infection. Subtotal resection of the stomachs of three H. pylori-infected and asymptomatic obese patients were collected after a sleeve gastrectomy. Distribution and density of H. pylori were determined using culture and RT-PCR on multiple gastric sites (88, 176, and 101 biopsies per patient). Diversity of H. pylori strains was studied using antibiotic susceptibility testing, random amplified polymorphism DNA (RAPD) typing and cagA gene detection on single-colony isolates (44, 96, and 49 isolates per patient). H. pylori was detected in nearly all analyzed sites (354/365 biopsies, 97%). Antral density was higher in one patient only. The three stomachs were almost exclusively infected by an antibiotic-susceptible strain. One clarithromycin-resistant isolate in one biopsy was detected in two stomachs (1/44 and 1/49 isolates), while in the third one, eight (8/96 isolates) metronidazole-resistant isolates were detected. DNA typing showed infection with cagA-negative strains for one patient, cagA-positive strains for a second patient and the third patient was infected with two different strains of distinct cagA genotypes. Infection with H. pylori is shown to spread to the whole surface of the stomach, but a possibility of minor sub-population of antibiotic-resistant clones, undetectable in routine practice.

Diagnostic Accuracy of a Noninvasive Test for Detection of Helicobacter pylori and Resistance to Clarithromycin in Stool by the Amplidiag H. pylori+ClariR Real-Time PCR Assay.

The noninvasive detection of Helicobacter pylori and its resistance to clarithromycin could revolutionize the management of H. pylori-infected patients by tailoring eradication treatment without any need for endoscopy when histology is not necessary. Several real-time PCR tests performed on stools have been proposed, but their performances were either poor or they were tested on too few patients to be properly evaluated. We conducted a prospective, multicenter study including 1,200 adult patients who were addressed for gastroduodenal endoscopy with gastric biopsies and who were naive for eradication treatment in order to evaluate the performance of the Amplidiag H. pylori+ClariR assay recently developed by Mobidiag (Espoo, Finland). The results of the Amplidiag H. pylori+ClariR assay performed on DNA from stools (automatic extraction with the EasyMag system [bioMérieux]) were compared with those of culture/Etest and quadruplex real-time PCRs performed on two gastric biopsy samples (from the antrum and corpus) to detect the H. pyloriglmM gene and mutations in the 23S rRNA genes conferring clarithromycin resistance. The sensitivity and specificity of the detection of H. pylori were 96.3% (95% confidence interval [CI], 92 to 98%) and 98.7% (95% CI, 97 to 99%), respectively. The positive and negative predictive values were evaluated to be 92.2% (95% CI, 92 to 98%) and 99.3% (95% CI, 98 to 99%), respectively. In this cohort, 160 patients (14.7%) were found to be infected (positive by culture and/or PCR). The sensitivity and specificity for detecting resistance to clarithromycin were 100% (95% CI, 88 to 100%) and 98.4% (95% CI, 94 to 99%), respectively.

First Detection in Helicobacter suis of a Mutation Conferring Resistance to Clarithromycin in Helicobacter pylori: Case Report and Review of the Literature.

Helicobacter pylori and non-H. pylori Helicobacter (NHPH) are associated with gastritis, ulcer, and gastric neoplasia. Because of the impossibility to culture them, diagnosis remains based on microscopic examination and molecular analysis of biopsies. Owing to the lack of data concerning antibiotic resistance of NHPH, infected patients are usually treated using antibiotics, including clarithromycin. Herein, we describe, for the first time a human infection by Helicobacter suis harboring a mutation associated to clarithromycin resistance in H. pylori. Eradication was successful with a metronidazole-based treatment. This observation highlights the benefit to use genotypic detection of resistance to improve therapeutic management of NHPH infections.

Th-17 response and antimicrobial peptide expression are uniformly expressed in gastric mucosa of Helicobacter pylori-infected patients independently of their clinical outcomes.

BACKGROUND: The pathological determinism of H. pylori infection is explained by complex interplay between bacterial virulence and host inflammatory response. In a large prospective multicenter clinical study, Th17 response, expression of antimicrobial peptides (AMPs), cagA and vacA status, and bacterial density were investigated in the gastric mucosa of H. pylori -infected patients. MATERIALS AND METHODS: Gastric inflammatory response was analyzed by RT-qPCR for quantification of Th17 cytokines (IL-17A, IL-22), CXCL-8, and AMPs (BD2 and S100A9) mRNA levels in gastric biopsies. Detection and genotyping of H. pylori strains were achieved by bacterial culture and PCR. RESULTS: Among 787 patients screened for H. pylori, 269 were analyzed (147 H. pylori -infected and 122 uninfected patients). In H. pylori -infected patients, distribution was 83 gastritis, 12 duodenal ulcers, 5 gastric ulcers, and 47 precancerous and cancerous lesions. CXCL-8, IL-17A, BD2, and S100A9 mRNA levels were significantly increased in H. pylori -infected patients but, surprisingly, IL-22 was not, and no difference was shown between H. pylori -related diseases. A positive correlation was identified between S100A9 expression and bacterial density. Although expression of the virulence genes cagA and vacA did not impact inflammatory response, patients infected with a cagA-positive strain were associated with severe H. pylori -related diseases. CONCLUSION: This study showed that CXCL-8, IL-17A, and AMPs are not differently expressed according to the various H. pylori -related diseases. The clinical outcome determinism of H. pylori infection is most likely not driven by gastric inflammation but rather tends to mainly influenced by bacterial virulence factors.

In vitro culture and phenotypic and molecular characterization of gastric stem cells from human stomach.

BACKGROUND: Human gastric mucosa shows continuous self-renewal via differentiation from stem cells that remain poorly characterized. METHODS: We describe an original protocol for culture of gastric stem/progenitor cells from adult human stomach. The molecular characteristics of cells were studied using TaqMan low-density array and qRT-PCR analyses using the well-characterized H1 and H9 embryonic stem cells as reference. Epithelial progenitor cells were challenged with H. pylori to characterize their inflammatory response. RESULTS: Resident gastric stem cells expressed specific molecular markers of embryonic stem cells (SOX2, NANOG, and OCT4), as well as others specific to adult stem cells, particularly LGR5 and CD44. We show that gastric stem cells spontaneously differentiate into epithelial progenitor cells that can be challenged with H. pylori. The epithelial progenitor response to H. pylori showed a cag pathogenicity island-dependent induction of matrix metalloproteinases 1 and 3, chemokine (CXCL1, CXCL5, CXCL8, CCL20) and interleukine 33 expression. CONCLUSION: This study opens new outlooks for investigation of gastric stem cell biology and pathobiology as well as host-H. pylori interactions.

Chemokines and antimicrobial peptides have a cag-dependent early response to Helicobacter pylori infection in primary human gastric epithelial cells.

Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.

Genetic analysis of glycopeptide-resistant Staphylococcus epidermidis strains from bone and joint infections.

Glycopeptide-resistant Staphylococcus epidermidis (GRSE) strains are of increasing concern in bone and joint infections (BJIs). Using multilocus sequence typing and multilocus variable-number tandem repeat analysis, we show that BJI-associated GRSE strains are genetically diverse but arise from related, multiresistant hospital sequence types (STs), mostly ST2, ST5, and ST23.

Distribution of spontaneous gyrA mutations in 97 fluoroquinolone-resistant Helicobacter pylori isolates collected in France.

We determined the prevalence of gyrA mutations conferring fluoroquinolone resistance in 97 Helicobacter pylori isolates collected in France from 2007 to 2010. Ninety-four harbored one or two mutations already found in the quinolone resistance determining region (QRDR) of gyrA (for T87I, n = 23; for N87K, n = 32; for D91N, n = 30; for D91G, n = 7; for D91Y, n = 6), 2 harbored a mutation never previously described (D91H and A88P), and one strain was resistant (ciprofloxacin MIC of 8 mg/liter) without a detected mutation conferring this resistance in gyrA or gyrB genes.

Decreased susceptibility to teicoplanin and vancomycin in coagulase-negative Staphylococci isolated from orthopedic-device-associated infections.

We studied 315 coagulase-negative Staphylococcus strains recovered prospectively during 240 surgical procedures (206 subjects) from proven or suspected device-associated bone and joint infections. Sixteen strains (5.1%) had decreased susceptibility to glycopeptides: 15 (12 S. epidermidis strains, 2 S. capitis strains, and 1 S. haemolyticus strain) to teicoplanin alone (MIC of 16 mg/liter, n = 9; MIC of 32 mg/liter, n = 6) and one (S. epidermidis) to both teicoplanin and vancomycin (MIC, 16 and 8 mg/liter, respectively). Decreased susceptibility to teicoplanin was more prevalent in "infecting" strains (i.e., strains recovered from >/=2 distinct intraoperative samples) than in "contaminants" (i.e., strains not fulfilling this criterion) (8.1% [12/149] versus 2.4% [4/166], respectively [P = 0.022]). One hundred percent (13/13) of S. epidermidis strains with decreased susceptibility to teicoplanin were resistant to methicillin (versus 112/173 [64.7%] for S. epidermidis strains susceptible to teicoplanin; P = 0.021).