Activation of class 1 integron integrase is promoted in the intestinal environment.

Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.

Breast milk in neonate oral care: oropharyngeal effects in extremely preterm infants.

Ventilator-associated pneumonia (VAP) is a frequent nosocomial infection in neonatal intensive care units (NICU). Extremely preterm infants are at highest risk of developing VAP. Several studies indicate that oral care included in a preventive protocol effectively reduces neonatal VAP incidence. We investigated the effects of oral care with breast milk on oral immune defenses and microbiota in extremely preterm infants. Thirty infants born ≤ 30 weeks gestation hospitalized at our NICU were selected and divided into three groups: oral care with breast milk, formula, or sterile water. Effects on oral immune defenses in vivo were studied using ELISA to measure lactoferrin (LF) and secretory immunoglobulin A (sIgA) in pharyngeal aspirates before and after oral care. Different LF concentrations were tested in vitro to assess their effects on loads of selected bacterial species by culture. Effects on selected bacteria potentially responsible for VAP in vivo were studied by real-time PCR detection in pharyngeal aspirates before and after oral care. Oral care with breast milk significantly increases LF concentrations to 69.8 × 103 ng/ml (p = 0.012) and sIgA to 36.8 × 103 ng/ml (p = 0.017) in vivo. These LF concentrations considerably reduce loads of E. coli, S. epidermidis, S. aureus, and P. aeruginosa, in vitro. However, contrary to our expectation, no effect on colonization of bacteria most commonly responsible for VAP was found in vivo. CONCLUSION: In extremely preterm infants, oral care with breast milk increases local immune defense markers (LF, sIgA), which combat bacterial infections. Further clinical trials should be conducted to evaluate their effects on VAP prevention in neonates. WHAT IS KNOWN: • The population at higher risk to develop VAP are preterm infants. • Several studies indicate oral care within a preventive bundle is effective in reducing neonatal VAP incidence. WHAT IS NEW: • In extremely premature infants, oral care with breast milk causes a significant increase in local immune defences in terms of lactoferrin (LF) and secretory immunoglobulin A (sIgA). • LF concentrations obtained after oral care with breast milk decreased loads of bacteria most commonly responsible for VAP in premature infants under experimental in-vitro.

Emergence and Within-Host Genetic Evolution of Methicillin-Resistant Staphylococcus aureus Resistant to Linezolid in a Cystic Fibrosis Patient.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has increased in recent years among cystic fibrosis (CF) patients. Linezolid (LZD) is one of the antistaphylococcal antibiotics widely used in this context. Although LZD resistance is rare, it has been described as often associated with long-term treatments. Thirteen MRSA strains isolated over 5 years from one CF patient were studied for LZD resistance emergence and subjected to whole-genome sequencing (WGS). Resistance emerged after three 15-day LZD therapeutic regimens over 4 months. It was associated with the mutation of G to T at position 2576 (G2576T) in all 5 rrl copies, along with a very high MIC (>256 mg/liter) and a strong increase in the generation time. Resistant strains isolated during the ensuing LZD therapeutic regimens and until 13 months after LZD stopped harbored only 3 or 4 mutated rrl copies, associated with lower MICs (8 to 32 mg/liter) and low to moderate generation time increases. Despite these differences, whole-genome sequencing allowed us to determine that all isolates, including the susceptible one isolated before LZD treatment, belonged to the same lineage. In conclusion, LZD resistance can emerge rapidly in CF patients and persist without linezolid selective pressure in colonizing MRSA strains belonging to the same lineage.

Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants.

OBJECTIVES: To establish the reference values, diagnostic accuracy, and effect of various factors on cell count in intubated preterm neonates subjected to nonbronchoscopic bronchoalveolar lavage. STUDY DESIGN: This prospective, cross-sectional, blinded study included preterm neonates ventilated for any reason who underwent nonbronchoscopic bronchoalveolar lavage if they had not previously received postnatal antibiotics or steroids. Lavage was performed before surfactant replacement, if any. A gentle ventilation policy was applied. Pneumonia was diagnosed using clinical criteria, without considering cell count. Investigators performing cell counts were blinded to the clinical data. RESULTS: There were 276 neonates enrolled; 36 had congenital or ventilator-associated pneumonia. In the 240 noninfected babies, median neutrophil count increased significantly after the first 2 days of ventilation (day 1, 2 cells per field [IQR, 0.0-9.5 cells per field]; day 2, 2 cells per field [IQR, 0-15 cells per field]; day 3, 20 cells per field [IQR, 2-99 cells per field]; day 4, 15 cells per field [IQR, 2-96 cells per field]; P < .0001). No significant difference was seen over time in infected babies. Multivariate analysis indicated pneumonia (standardized ß = 0.134; P = .033) and the time spent under mechanical ventilation before nonbronchoscopic bronchoalveolar lavage as factors significantly influencing neutrophil count (standardized ß = 0.143; P = .027). Neutrophil count was correlated with the duration of ventilation (rho = 0.28; P <.001). Neutrophil counts were higher in infected (24 cells/field [IQR, 5-78] cells/field) than in noninfected babies (4 cells/field [IQR, 1-24 cells/field]; P <.001) and had an moderate reliability for pneumonia within the first 2 days of ventilation (area under the curve, 0.745; (95% CI, 0.672-0.810; P = .002). CONCLUSIONS: We provide reference values for airway neutrophil counts in ventilated preterm neonates. Bronchoalveolar lavage neutrophils significantly increase after 2 days of ventilation. Neutrophil count has moderate accuracy to diagnose pneumonia, but only within the first 2 days of ventilation.

Benefits of Rapid Molecular Diagnosis of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections in Women Attending Family Planning Clinics.

We evaluated the benefits of on-demand systematic screening for Chlamydia trachomatis and Neisseria gonorrhoeae using the Xpert CT/NG assay in 589 women attending family planning clinics. The sexually transmitted infection prevalence was 16.5% with 15.1% C. trachomatis and 3.1% N. gonorrhoeae infections. The on-demand test allowed for a quicker management of patients at high risk for sexually transmitted infections.

Group A Streptococcus endometritis following medical abortion.

Medical abortion is not recognized as a high-risk factor for invasive pelvic infection. Here, we report two cases of group A Streptococcus (GAS; Streptococcus pyogenes) endometritis following medical abortions with a protocol of oral mifepristone and misoprostol.

Evaluation of the Cepheid Xpert GBS assay for rapid detection of group B Streptococci in amniotic fluids from pregnant women with premature rupture of membranes.

The Xpert GBS real-time PCR assay for the detection of group B streptococci (GBS) in antepartum screening samples was evaluated on amniotic fluid samples collected from 139 women with premature rupture of membrane at term. When any intrapartum positive result from the Xpert GBS or culture was considered a true positive, the sensitivities of the Xpert GBS and culture were 92.3% and 84.6%, respectively. This assay could enhance exact identification of candidates for intrapartum antibiotic prophylaxis.

Prevalence of mupirocin resistance among invasive coagulase-negative staphylococci and methicillin-resistant Staphylococcus aureus (MRSA) in France: emergence of a mupirocin-resistant MRSA clone harbouring mupA.

OBJECTIVES: Mupirocin is the cornerstone of decolonization regimens, a successful strategy to prevent healthcare-associated staphylococcal infections. Several recent studies have reported alarming results: (i) an extending reservoir of mupA, the ancestral mobile resistance gene, among coagulase-negative staphylococci (CoNS); (ii) the emergence of a new resistance gene (mupB); and (iii) a growing number of mupirocin-resistant methicillin-resistant Staphylococcus aureus (MRSA), including highly pathogenic clones. We performed a nationwide prospective study in France to detect such trends among invasive staphylococci. METHODS: Between October 2011 and February 2012, 367 MRSA and 708 CoNS invasive isolates were collected from 37 hospitals and analysed centrally. Mupirocin MICs were determined using the broth microdilution method. mupA/B PCR was performed for resistant isolates (MIC >1 mg/L). Genetic relatedness between mupirocin-resistant MRSA isolates was determined by PFGE analysis and related isolates were tested by microarray. RESULTS: Among MRSA isolates 2.2% (n = 8) were classified as mupirocin resistant; 1.4% (n = 5) showing low-level resistance (MIC ≤256 mg/L) and 0.8% (n = 3) high-level resistance (MIC >256 mg/L). Only the latter isolates carried mupA. A clonal relationship was identified between two mupA-negative MRSA from the same hospital and three mupA-positive MRSA from three distant towns; these three isolates belonged to the Lyon clone. Mupirocin resistance was identified in 10.3% of CoNS, mainly highly resistant mupA-positive isolates (5.6%). The mupB gene was not detected in mupirocin-resistant MRSA or CoNS. CONCLUSIONS: This first large national study indicates the need for thorough epidemiological monitoring and a stewardship programme to prevent and detect mupirocin resistance in staphylococci.

Impact of SARS-CoV-2 Alpha and Gamma Variants among Symptomatic Pregnant Women: A Two-Center Retrospective Cohort Study between France and Brazil.

New variants of SARS-CoV-2 are a major source of concern, especially for pregnant women and in the perinatal context. The primary aim of this study was to compare the severity of COVID-19 infection in pregnant women depending on strain predominance between wild-type Alpha and Gamma variants. The secondary aim was to study the impact of these strains on obstetrical and neonatal outcomes. We conducted a two-center international retrospective cohort study, which included two type III maternity hospitals, one in France and one in Brazil, comparing the first period corresponding to the wild-type strain and the second period corresponding to the predominance of the Alpha variant in France and the Gamma variant in Brazil. We included 151 pregnant women with symptomatic SARS-CoV-2 infection confirmed by RT-PCR. The rate of severe-to-critical infection, according to the WHO definition, was seven-fold higher in patients infected during the "variant period" than in patients infected during the "wild-type period" (aOR = 7.07, 95CI [2.50−21.6], p < 0.001). There were no statistical differences concerning composite obstetrical and neonatal outcomes between the different periods. While analyzing each variant separately, it was found that, in France, the risk of developing a severe-to-critical infection was three times greater during the Alpha period than during the wild-type period (OR = 3.25, 95CI [0.70−15.6], p = 0.13) and, in Brazil, the risk was twelve times greater during the Gamma period than during the wild-type period (OR = 11.8, 95CI [2.46−72.3], p = 0.003). The Alpha and Gamma variants of SARS-CoV-2 seem to be more dangerous in the obstetrical context. With the rapid emergence of new variants, it is necessary to accelerate vaccination to protect women and newborn children.

Contaminated Incubators: Source of a Multispecies Enterobacter Outbreak of Neonatal Sepsis.

The genus Enterobacter includes species responsible for nosocomial outbreaks in fragile patients, especially in neonatal intensive care units (NICUs). Determining the primary source of infection is critical to outbreak management and patient outcomes. In this investigation, we report the management and control measures implemented during an Enterobacter outbreak of bloodstream infections in premature babies. The study was conducted in a French NICU over a 3-year period (2016 to 2018) and included 20 premature infants with bacteremia. The clinical and microbiological characteristics were identified, and whole-genome sequencing (WGS) was performed on bacteremia isolates. Initially, several outbreak containment strategies were carried out with no success. Next, outbreak investigation pinpointed the neonatal incubators as the primary reservoir and source of contamination in this outbreak. A new sampling methodology during "on" or "in use" conditions enabled its identification, which led to their replacement, thus resulting in the containment of the outbreak. WGS analysis showed a multiclonal outbreak. Some clones were identified in different isolation sources, including patients and neonatal incubators. In addition, microbiological results showed a multispecies outbreak with a high prevalence of Enterobacter bugandensis and Enterobacter xiangfangensis. We conclude that the NICU health care environment represents an important reservoir for Enterobacter transmission and infection. Finally, extracting samples from the neonatal incubator during active use conditions improves the recovery of bacteria from contaminated equipment. This method should be used more frequently to achieve better monitoring of the NICU for HAIs prevention. IMPORTANCE Neonatal incubators in the NICU can be an important reservoir of pathogens responsible for life-threatening outbreaks in neonatal patients. Traditional disinfection with antiseptics is not sufficient to eradicate the microorganisms that can persist for long periods in the different reservoirs. Identification and elimination of the reservoirs are crucial for outbreak prevention and control. In our investigation, using a new strategy of microbiological screening of neonatal incubators, we demonstrated that these were the primary source of contamination. After their replacement, the outbreak was controlled. This new methodology was effective in containing this outbreak and could be a viable alternative for infection prevention and control in outbreak situations involving incubators as a reservoir.

dnaJ: a New Approach to Identify Species within the Genus Enterobacter.

The taxonomy of the genus Enterobacter can be confusing and has been considerably revised in recent years. We propose a PCR and amplicon sequencing technique based on a partial sequence of the dnaJ gene for species assignment consistent with DNA-DNA digital hybridization (dDDH) and pairwise average nucleotide identity (ANI). We performed a validation of the method by comparing the type strains of each species, sequences obtained from the GenBank database, and clinical specimens. Our results show that the polymorphism of the target sequence of dnaJ allows the identification of species. Using this gene, we assigned the species to 100 strains deposited in the GenBank database that were consistent with the species assignment by dDDH and ANI. The analysis showed that using the partial dnaJ sequence is congruent with WGS as far as correct identification of Enterobacter species is concerned. Finally, we applied our dnaJ method on a national collection of 68 strains identified as Enterobacter isolated from the blood cultures of premature babies using an algorithm based on a type-strain library and the SeqScape software. For the first time, we identified Enterobacter quasihormaechei in blood cultures from four neonatal sepsis cases. We also noticed a higher prevalence of E. bugandensis (36.3%; 32/88) and E. xiangfangensis (46.5%; 41/88). E. bugandensis is a novel species recently described specifically in instances of neonatal sepsis. In conclusion, sequencing a part of the dnaJ gene could be a quick, more economical, and highly discriminating method of identifying Enterobacter species in clinical practice and research. IMPORTANCE We propose a new approach for Enterobacter species identification based on the diversity of the gene encoding the heat shock protein DnaJ. This new tool can be easily implemented in clinical laboratories in addition to identification by MALDI-TOF.

Improving tuberculosis management in prisons: Impact of a rapid molecular point-of-care test.

OBJECTIVE: To improve tuberculosis (TB) diagnosis in prison, we evaluate the value of the XpertⓇMTB/RIF Ultra assay (Xpert) as point-of-care (POC) in a French prison hospital. METHODS: We first validated Xpert use on raw sputum at the referent laboratory. Secondly, trained physicians at the prison hospital performed Xpert tests for each patient presenting TB symptoms. The results were compared with Xpert, microscopic examination, culture and drug susceptibility testing on the corresponding decontaminated specimens. RESULTS: 76 inmates were included in 15 months and 21 were diagnosed with TB. The overall sensitivity, specificity, positive and negative predictive values of Xpert were respectively: 92.3%, 100%, 100% and 98.7% on raw sputum. The efficiency of the molecular POC was confirmed by a concordance of 97% between Xpert findings from the prison hospital and culture results. Delay of microbiological diagnosis was reduced by about 18 days for 13 inmates with smear-negative sputum that avoid the mobilization of major means (escort, transport) to perform fibroscopic samples. Repeated Xpert negative results helped to speed the lifting of inmate isolation. CONCLUSIONS: The implementation of Xpert in prison could optimize the management of incarcerated patients and thus limit the spread of TB among inmates, carers and other staff.

Presence of 2-hydroxymyristate on endotoxins is associated with death in neonates with Enterobacter cloacae complex septic shock.

Enterobacter cloacae complex species are involved in infections among critically ill patients. After a recent E.cloacae outbreak of fulminant neonatal septic shock, we conducted a study to determine whether septic shock severity and its lethal consequence are related to structural features of the endotoxin (lipopolysaccharide [LPS]) of the strains isolated from hospitalized infants and more specifically its lipid A region. It appeared that the LPSs are very heterogeneous, carrying fifteen different molecular species of lipid A. The virulence was correlated with a structural feature identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and gas chromatography coupled with mass spectrometry: the presence of 2-hydroxymyristic acid as a secondary substituent in lipid A. This is the first published evidence linking LPS structural moiety to neonatal sepsis outcome and opens the possibility of using this fatty acid marker as a detection tool for high-risk patients, which could help reduce their mortality.

Post lockdown COVID-19 seroprevalence and circulation at the time of delivery, France.

BACKGROUND: To fight the COVID-19 pandemic, lockdown has been decreed in many countries worldwide. The impact of pregnancy as a severity risk factor is still debated, but strict lockdown measures have been recommended for pregnant women. OBJECTIVES: To evaluate the impact of the COVID-19 pandemic and lockdown on the seroprevalence and circulation of SARS-CoV-2 in a maternity ward in an area that has been significantly affected by the virus. STUDY DESIGN: Prospective study at the Antoine Béclère Hospital maternity ward (Paris area, France) from May 4 (one week before the end of lockdown) to May 31, 2020 (three weeks after the end of lockdown). All patients admitted to the delivery room during this period were offered a SARS-CoV-2 serology test as well concomitant SARS-CoV-2 RT-PCR on one nasopharyngeal sample. RESULTS: A total of 249 women were included. Seroprevalence of SARS-CoV-2 was 8%. The RT-PCR positive rate was 0.5%. 47.4% of the SARS-CoV-2-IgG-positive pregnant women never experienced any symptoms. A history of symptoms during the epidemic, such as fever (15.8%), myalgia (36.8%) and anosmia (31.6%), was suggestive of previous infection. CONCLUSIONS: Three weeks after the end of French lockdown, SARS-CoV-2 infections were scarce in our region. A very high proportion of SARS-CoV-2-IgG-negative pregnant women, which is comparable to that of the general population, must be taken into consideration in the event of a resurgence of the pandemic. The traces of a past active circulation of the virus in this fragile population during the spring wave should encourage public health authorities to take specific measures for this independent at-risk group, in order to reduce viral circulation in pregnant patients.

Proficiency program for real-time PCR diagnosis of Bordetella pertussis infections in French hospital laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses.

With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/microl (0.2 to 2 CFU/microl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.

Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis.

Detection of the most frequently bacteria involved in prosthetic joint infection (PJI) is usually performed by conventional cultures. We report a case of early PJI due to Ureaplasma urealyticum, diagnosed by 16S rRNA gene sequence analysis, which highlights the interest of molecular methods if fastidious bacteria are involved in PJI.

Campylobacter bacteremia: clinical features and factors associated with fatal outcome.

BACKGROUND: Campylobacter bacteremia is uncommon. The influence of underlying conditions and of the impact of antibiotics on infection outcome are not known. METHODS: From January 2000 through December 2004, 183 episodes of Campylobacter bacteremia were identified in 23 hospitals in the Paris, France, area. The medical records were reviewed. Characteristics of bacteremia due to Campylobacter fetus and to other Campylobacter species were compared. Logistic regression analysis was performed to identify risk factors for fatal outcome within 30 days. RESULTS: Most affected patients were elderly or immunocompromised. C. fetus was the most commonly identified species (in 53% of patients). The main underlying conditions were liver disease (39%) and cancer (38%). The main clinical manifestations were diarrhea (33%) and skin infection (16%). Twenty-seven patients (15%) died within 30 days. Compared with patients with bacteremia due to other Campylobacter species, patients with C. fetus bacteremia were older (mean age, 69.5 years vs. 55.6 years; P = .001) and were more likely to have cellulitis (19% vs. 7%; P = .03), endovascular infection (13% vs. 1%; P = .007), or infection associated with a medical device (7% vs. 0%; P = .02). Independent risk factors for death were cancer (odds ratio [OR], 5.1; 95% confidence interval [CI], 1.2-20.8) and asymptomatic infection (OR, 6.7; 95% CI, 1.5-29.4) for C. fetus bacteremia, the absence of prescription of appropriate antibiotics (OR, 12.2; 95% CI, 0.9-157.5), and prescription of third-generation cephalosporins (OR, 10.2; 95% CI, 1.9-53.7) for bacteremia caused by other species. CONCLUSIONS: Campylobacter bacteremia occurs mainly in immunocompromised patients. Clinical features and risk factors of death differ by infection species.

Primary peritonitis due to Streptococcus pyogenes with reduced susceptibility to fluoroquinolones.

We report the 1st case of primary peritonitis caused by Streptococcus pyogenes with a reduced susceptibility to fluoroquinolones in a healthy woman, leading to a clinical and microbiological treatment failure. DNA sequencing of parC and gyrA revealed the association of the following 3 substitutions in ParC: Ser80Pro, Asp91Asn, and Ser140Pro.

[Establishment of the intestinal microflora in neonates]. / Mise en place de la flore intestinale du nouveau-né

The intestinal microbiota is a complex ecosystem harbouring about 10(14) micro-organisms composed of nearly 400 hundred species. It plays various important functions in the gut, including metabolic, flora, barrier and stimulation of the intestinal immune system. Most studies have been based on culture, but more recent molecular biology techniques have provided complementary information. The formation of this ecosystem begins rapidly in the newborn; it is sterile at birth and is based on contact with the maternal flora and the surrounding environment. Although little is known about the factors leading to the development of bacteria, numerous external factors will affect the microbial succession: mode of delivery, environmental conditions, type of feeding, gestational age, and antibiotics. Recent data report a delay in intestinal colonization especially of enteric maternal bacteria. Which may be due to strict hygiene measures during birth. The clinical impact of these variations is not known but they could lead to lack of barrier flora or poor immune system stimulation in the gut. Modulation of gut microbiota in neonates with infant formulas containing either probiotics, prebiotics or non viable bacterias and their metabolites, or nucleotides is discussed.