Using MALDI-TOF MS typing method to decipher outbreak: the case of Staphylococcus saprophyticus causing urinary tract infections (UTIs) in Marseille, France.

Staphylococcus saprophyticus is one of the leading causes of urinary tract infections (UTI). In December 2014, our surveillance system identified an abnormal increase in S. saprophyticus causing UTIs in four university hospitals in Marseille, indicating a suspected community S. saprophyticus UTI outbreak. This was detected by our surveillance system BALYSES (Bacterial real-time Laboratory-based Surveillance System). S. saprophyticus/ Escherichia coli UTI ratio increased three-fold from 0.0084 in 2002 to 0.025 in December 2015 in Marseille with an abnormal peak in December 2014, and with an annual estimated ratio trend of 5.10-6 (p-value < 10-3). Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) spectral analysis of strains was used to analyse strains cluster expansion, comparing strains from Marseille to those from Nice during the same period. MALDI-TOF MS spectral analysis revealed a geographical restricted clonal expansion of the strains clusters in Marseille as compared to Nice. Our finding suggests (i) a geographically restricted expansion of a specific S. saprophyticus strain clusters circulating in Marseille, and (ii) MALDI-TOF MS can be used as a cost-effective tool to investigate an outbreak.

Dramatic decrease of Streptococcus pneumoniae infections in Marseille, 2003-2014.

We studied the evolution of the prevalence of pneumococcal infections in university hospitals in Marseille, France, from January 2003 to December 2014, and compared our observations and results to available international data. We collected data referring to patients hospitalised for Streptococcus pneumoniae infections in the four university hospitals of Marseille from January 2003 to December 2014. We then calculated percentages of positiveness to pneumococcal strains by dividing the annual number of patients infected by pneumococcal strains by the annual number of patients found to be infected by at least one bacterial species in the settings of interest throughout the study period. Overall, 2442 non-redundant patients were infected by S. pneumoniae strains throughout the study period. We observed that the annual percentage of patients infected by S. pneumoniae significantly decreased throughout the study period (from 1.99% in 2003 to 0.77% in 2014, p-value < 10(-4)). A significant correlation was obtained comparing the annual evolution of the percentage of patients positive to pneumococcal strains aged under 21 years to that of patients aged over 21 years (r = 0.93, p-value < 10(-5)). Our results allowed us to prove that national immunisation programmes effectively impact on the pneumococcal infection prevalence in young and elderly populations, even on the regional scale.

Evaluation of the PREVI® Isola automated seeder system compared to reference manual inoculation for antibiotic susceptibility testing by the disk diffusion method.

The disk diffusion (DD) method remains the most popular manual technique for antibiotic susceptibility testing (AST) in clinical microbiology laboratories. This is because of its simplicity, reproducibility, and limited cost compared to (automated) microdilution systems, which are usually less sensitive at detecting certain important mechanisms of resistance. Here, we evaluate the PREVI® Isola automated seeder system using a new protocol for spreading bacterial suspensions (eight deposits of calibrated inocula of bacteria, followed by two rounds of rotation) in comparison with manual DD reference testing on a large series of clinical and reference strains. The average time required for seeding one agar plate for DD with this new protocol was 51 s per plate, i.e., 70 agar plates/h. Reproducibility and repeatability was assessed on three reference and three randomly chosen clinical strains, as usually requested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and was excellent compared to the manual method. The standard deviations of zones of growth inhibition showed no statistical discrimination. The correlation between the two methods, assessed using 294 clinical isolates and a panel of six antibiotics (n = 3,528 zones of growth inhibition measured), was excellent, with a correlation coefficient of 0.977. The new PREVI® Isola protocol adapted for DD had a sensitivity of 99 % and a specificity of 100 % compared to the manual technique for interpreting DD as recommended by the EUCAST.

Molecular epidemiology and distribution of serotypes, genotypes, and antibiotic resistance genes of Streptococcus agalactiae clinical isolates from Guelma, Algeria and Marseille, France.

This study describes, for the first time, the genetic and phenotypic diversity among 93 Streptococcus agalactiae (group B Streptococcus, GBS) isolates collected from Guelma, Algeria and Marseille, France. All strains were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The molecular support of antibiotic resistance and serotyping were investigated by polymerase chain reaction (PCR). The phylogenetic lineage of each GBS isolate was determined by multilocus sequence typing (MLST) and grouped into clonal complexes (CCs) using eBURST. The isolates represented 37 sequence types (STs), 16 of which were novel, grouped into five CCs, and belonging to seven serotypes. Serotype V was the most prevalent serotype in our collection (44.1%). GBS isolates of each serotype were distributed among multiple CCs, including cps III/CC19, cps V/CC1, cps Ia/CC23, cps II/CC10, and cps III/CC17. All isolates presented susceptibility to penicillin, whereas resistance to erythromycin was detected in 40% and tetracycline in 82.2% of isolates. Of the 37 erythromycin-resistant isolates, 75.7% showed the macrolide-lincosamide-streptogramin B (MLSB)-resistant phenotype and 24.3% exhibited the macrolide (M)-resistant phenotype. Constitutive MLSB resistance (46%) mediated by the ermB gene was significantly associated with the Guelma isolates, whereas the M resistance phenotype (24.3%) mediated by the mefA/E gene dominated among the Marseille isolates and belonged to ST-23. Tetracycline resistance was predominantly due to tetM, which was detected alone (95.1%) or associated with tetO (3.7%). These results provide epidemiological data in these regions that establish a basis for monitoring increased resistance to erythromycin and also provide insight into correlations among clones, serotypes, and resistance genes.

Evaluation of colistin susceptibility in multidrug-resistant clinical isolates from cystic fibrosis, France.

The emergence of multidrug-resistant (MDR) bacteria in cystic fibrosis (CF) patients has led to the use of colistin drug and the emergence of colistin-resistant Gram-negative bacteria. The aim of this study was to compare the disk diffusion and Etest methods for colistin susceptibility testing on MDR bacteria associated with CF from Marseille, France. Forty-nine MDR clinical isolates (27 Stenotrophomonas maltophilia, 22 Achromobacter xylosoxidans) were used in this study. Disk diffusion and Etest assays were used to assess the reliability of these two techniques. For S. maltophilia, 25 out of 27 isolates had low minimum inhibitory concentrations (MICs, 0.125-0.75 mg/L), whereas two isolates displayed high MICs (32 mg/L). Similarly, 19 out of 22 A. xylosoxidans isolates had low MICs (0.75-3.0 mg/L), whereas three isolates had high MICs (32-256 mg/L). The diameters of zone inhibition with a 50-µg colistin disk displayed a good correlation with the MICs obtained by the Etest. Susceptible and resistant strains were eventually separated using a disk diffusion assay at a cut-off of ≤ 12 mm for a 50-µg disk. Colistin displayed excellent activity against S. maltophilia and A. xylosoxidans and the disk diffusion assay could be confidently used to determine the susceptibility to colistin for MDR Gram-negative bacteria in the context of CF.

Q fever and pregnancy: disease, prevention, and strain specificity.

The link between fetal morbidity and Q fever and the necessity of long-term antibiotics for Coxiella burnetii infection during pregnancy have been recently questioned in the Netherlands, where the clone responsible for the Q fever outbreak harbors the QpH1 plasmid. In this context, we assessed pregnancy outcomes according to antibiotic administration in a new series and compared the plasmid type between isolates associated with abortion and other clinical isolates to determine if there is a link between genotype and abortion in humans. All French patients who received a diagnosis of Q fever during pregnancy at the French National Referral Centre for Q Fever from 2006 through July 2011 were included. On the other hand, the plasmid types of 160 clinical isolates, including seven isolates from patients who experienced an abortion, were compared. The differences between the QpDV and QpH1 plasmid sequences were analyzed. Acute Q fever was a cause of fetal morbidity, and the absence of long-term cotrimoxazole therapy was associated with fetal death (p < 0.0001). Genotypic analysis showed that the QpDV plasmid was more frequent in isolates associated with abortion (p = 0.03). A comparison of the plasmid sequences revealed that four QpDV proteins had no direct counterparts in QpH1, with two whose functions were not present in QpH1. The different obstetrical morbidity of C. burnetii relative to different geographical areas could be related to strain specificity, possibly based on differences in plasmid sequences, or to a failure of public health authorities to detect early miscarriages.

Staphylococcal cassette chromosome mec characterization of methicillin-resistant Staphylococcus aureus strains isolated at the military hospital of Constantine/Algeria.

Staphylococcal cassette chromosome mec is a genetic mobile element that carries the gene mecA mediating the methicillin resistance in staphylococci. The aim of this study is to type the Staphylococcal cassette chromosome mec (SCCmec) in 64 non-redundant methicillin-resistant Staphylococcus aureus (MRSA) strains recovered at the military hospital of Constantine (Algeria) between 2005 and 2007. Methicillin resistance was detected by oxacillin and cefoxitin discs and PBP2a test, and then confirmed by mecA PCR. The SCCmec complex types were determined by real time PCR. The analysis showed that 50 isolates were hospital acquired (HA-MRSA) and 14 were community-acquired (CA-MRSA). SCCmec type IV and V (traditionally attributed to CA-MRSA) were harbored by both HA-MRSA and CA-MRSA, while SCCmec type I, II and III were not recorded. These findings motivate more investigations to be carried on HA-MRSA in our hospital and other national health care centers.

First detection of vanA positive Enterococcus faecium clonal complex 17 in hospital wastewater in Algeria: an epidemiological report.

Enterococcus spp. are Gram-positive cocci that are recognised as critical opportunistic pathogens, especially in immunocompromised patients. Vancomycin is considered as the drug of last resort for the treatment of infections caused by Enterococcus species, making vancomycin resistance a serious public health concern. In this article, we report the first environmental vanA positive Enterococcus faecium isolates in Algeria. The strains were selectively isolated from hospital wastewater and then identified using matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry. Antibiotic susceptibility testing was performed using the disc diffusion method. Vancomycin resistance genes were searched for by standard PCR and the clonal relatedness of our isolates was investigated by multilocus sequence typing. A total of five highly vancomycin-resistant Gram-positive bacteria were isolated and identified as Enterococcus faecium. The isolates harboured the vanA gene and were assigned to the clonal complex 17. Our findings confirm the great potential of hospital wastewater as a reservoir and dissemination pathway of multidrug resistant organisms, and alert to the need for better regulation of hospital waste management in order to reduce their impact on the environment.

Evidence of circulation of an epidemic strain of Francisella tularensis in France by multispacer typing.

Multispacer typing (MST) was used to type ten Francisella tularensis strains detected in French patients. Incorporating 79 Swedish F. tularensis strains, phylogenetic analysis demonstrated that although tularemia appears as a sporadic disease in France, it is caused by an epidemic cluster of strains.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates in Algeria.

INTRODUCTION: The epidemiology of Staphylococcus aureus has changed radically since 1999, in particular, methicillin-resistant S. aureus (MRSA), originally restricted to hospital, has emerged as a significant pathogen in the community, and true community-acquired MRSA (CA-MRSA) infections have been reported in patients with no clear risk factors. CA-MRSA strains frequently produce Panton-Valentine leukocidin (PVL). OBJECTIVES: The objectives of this study were: (i) to monitor the prevalence of PVL and toxic shock syndrome toxin-1 (TSST-1) isolates MRSA; (ii) to identify the staphylococcal cassette chromosome (SCCmec) types of MRSA isolates. MATERIAL AND METHODS: Sixty-four isolates, collected between 2005 and 2007 in Didouche Mourad hospital of Algeria. The isolates were identified by conventional methods. The antibiotic susceptibility of the isolates was performed using the disk diffusion method and automat Vitek2. The presence of gene mecA, the genes encoding SCCmec type, PVL and TSST-1 toxins were investigated by real-time PCR. RESULTS: All strains were gene mecA positives, 32 (50%) harboured SCCmec IV type, 28 (43.75%) harboured SCCmec V type. 19 (29.68%) have been identified positive for the leukocidin toxin (PVL), they harboured SCCmec type IV. The virulence factor TSST-1 was not present among these isolates. CONCLUSION: These results show a high prevalence of PVL-positive H-MRSA in our wards.

Genome analysis of Lachnoclostridium phocaeense isolated from a patient after kidney transplantation in Marseille.

Lachnoclostridium phocaeense is a new species in the genus Lachnoclostridium. Lachnoclostridium phocaeense is a Gram-positive anaerobic rod. This strain, Marseille-P3177T (CSUR = P3177) with the below described genome was isolated from the urine sample of a women after kidney transplantation. The strain genome is 3 500 754 bp long with 50.62% G + C content and consists of a single contig (GenBank accession number NZ_LT635479.1).

Seroprevalence of Q fever in a district located in the west Black Sea region of Turkey.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. In Turkey, it has been reported from the late 1940s that Q fever is endemic in humans and animals. Our objective was to evaluate the seroprevalence in Samsun Tekkeköy (north Turkey), where an outbreak of Q fever occurred in 2002. In this cross-sectional study, subjects were selected by the random proportional sampling method. All subjects were healthy with no specific symptoms and tested by the microimmunofluorescent antibody test. In total, we tested 407 subjects; 33 (8.1%) of them were identified as past evidence of infection and 22 (5.4%) were considered as evolutive form of Q fever (17 acute and five chronic forms). The seroprevalence was significantly higher among people over 30 years of age, hunters, and slaughters than the others (p = 0.001, p = 0.034, and p = 0.006, respectively). We found 13.5% seropositivity among healthy subjects, confirming that Q fever is prevalent in our region and is often asymptomatic.

What could explain the late emergence of COVID-19 in Africa?

At the end of November 2019, a novel coronavirus responsible for respiratory tract infections emerged in China. Despite drastic containment measures, this virus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread in Asia and Europe. The pandemic is ongoing with a particular hotspot in southern Europe and America in spring 2020. Many studies predicted an epidemic in Africa similar to that currently seen in Europe and the USA. However, reported data do not confirm these predictions. Several hypotheses that could explain the later emergence and spread of the coronavirus disease 2019 (COVID-19) pandemic in African countries are being discussed, including the lack of health-care infrastructure capable of clinically detecting and confirming COVID-19 cases, the implementation of social distancing and hygiene, international air traffic flows, the climate, the relatively young and rural population, the genetic polymorphism of the angiotensin-converting enzyme 2 receptor, cross-immunity and the use of antimalarial drugs.

Clinical efficacy of chloroquine derivatives in COVID-19 infection: comparative meta-analysis between the big data and the real world.

In the context of the current coronavirus disease 2019 (COVID-19) pandemic, we conducted a meta-analysis on the effects of chloroquine derivatives in patients, based on unpublished and published reports available publicly on the internet as of 27 May 2020. The keywords 'hydroxychloroquine', 'chloroquine', 'coronavirus', 'COVID-19' and 'SARS-Cov-2' were used in the PubMed, Google Scholar and Google search engines without any restrictions as to date or language. Twenty studies were identified involving 105 040 patients (19 270 treated patients) from nine countries (Brazil, China, France, Iran, Saudi Arabia, South Korea, Spain and the USA). Big data observational studies were associated with conflict of interest, lack of treatment dosage and duration, and absence of favourable outcome. Clinical studies were associated with favourable outcomes and details on therapy. Among clinical studies, three of four randomized controlled trials reported a significant favourable effect. Among clinical studies, a significant favourable summary effect was observed for duration of cough (OR 0.19, p 0.00003), duration of fever (OR 0.11, p 0.039), clinical cure (OR 0.21, p 0.0495), death (OR 0.32, p 4.1 × 10-6) and viral shedding (OR 0.43, p 0.031). A trend for a favourable effect was noted for the outcome 'death and/or intensive care unit transfer' (OR 0.29, p 0.069) with a point estimate remarkably similar to that observed for death (∼0.3). In conclusion, a meta-analysis of publicly available clinical reports demonstrates that chloroquine derivatives are effective to improve clinical and virological outcomes, but, more importantly, they reduce mortality by a factor of 3 in patients with COVID-19. Big data are lacking basic treatment definitions and are linked to conflict of interest. The retraction of the only big data study associated with a significantly deleterious effect the day after (June 5, 2020) the acceptance of the present work (June 4, 2020) confirms the relevance of this work.

Phenotypic and genotypic characterization of clinical carbapenem-resistant Enterobacteriaceae isolates from Sokoto, northwest Nigeria.

Emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) are two of the major problems currently threatening global public health. In Nigeria, interest in CPE is recent. In Sokoto, northwest Nigeria, there are no data on the prevalence and mechanism underlying carbapenem resistance. In this study, we aimed to investigate the presence of clinical carbapenems-resistant Enterobacteriaceae isolates in two leading hospitals in Sokoto, northwest Nigeria. A total of 292 non-duplicate Enterobacteriaceae isolated from clinical specimens processed in the diagnostic laboratories of two hospitals between January and June 2019 were collected. Of these, 129 (44.2 %) and 19 (6.5%) were resistant to third-generation cephalosporin and carbapenems, respectively. RT-PCR revealed that 10 (7.8%), 19 (14.7%) and 46 (35.7%) of the third-generation cephalosporin-resistant isolates harboured bla SHV, bla TEM and bla CTX-M genes, respectively. The modified Carba NP test result showed that only 7 (36.8 %) of the 19 carbapenem-resistant isolates were carbapenemase producing; among them, bla NDM-5 and bla OXA-181 genes were identified in five and two isolates, respectively. However, none of the carbapenemase genes investigated, including bla VIM, bla KPC and bla IMP, was detected in the remaining carbapenem-resistant isolates, suggesting a non-enzymatic mechanism. This study reports for the first time, the emergence of CPE in Sokoto state and the detection of NDM-producing Citrobacter freundii in Nigeria. The observed CPE in this study is a concern in a country where alternative antibiotics are rarely available.

Real-time PCR strategy and detection of bacterial agents of lymphadenitis.

The aim of this study was to compare 16 S rRNA gene amplification and sequencing with a systematic real-time PCR assay screening strategy that includes all common known pathogens recovered from lymph node biopsy specimens. Lymph node biopsy samples sent to our laboratory from January 2007 to December 2008 were tested in the study. Lymph nodes were screened for the presence of any bacteria by PCR amplification and sequencing targeting the 16 S rRNA gene and also by a specific real-time PCR strategy that includes Bartonella henselae, mycobacteria, Francisella tularensis, and Tropheryma whipplei. By testing 491 lymph nodes, we found that the sensitivity of our specific real-time PCR assay strategy was significantly higher than 16 S rRNA PCR amplification and sequencing for the detection of Bartonella henselae (142 vs 98; p < 10(-4)), Francisella tularensis (16 vs 10, p < 10(-4)), and mycobacteria (8 versus 3, p < 10(-4)). None of the samples was positive for Tropheryma whipplei. Our study demonstrates the usefulness and specificity of a systematic real-time PCR strategy for molecular analysis of lymph node biopsy specimens and the higher sensitivity compared with standard 16 S rRNA gene amplification and sequencing.

Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing.

Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

Emergence of plasmid-encoded VIM-2-producing Pseudomonas aeruginosa isolated from clinical samples in Lebanon.

The present study aimed to describe the emergence of carbapenem-resistant Pseudomonas aeruginosa isolated from clinical Lebanese patients. The resistance of these isolates is due to the presence of the plasmid-encoded bla VIM-2 gene. We provide its first description in Lebanon, as well as a description of disruption of the oprD gene by mutations.

Erratum to "Antibacterial Activity of Chalcone and Dihydrochalcone Compounds from Uvaria chamae Roots against Multidrug-Resistant Bacteria".

[This corrects the article DOI: 10.1155/2018/1453173.].

Detection of new genotypes of Orientia tsutsugamushi infecting humans in Thailand.

PCR screening of blood specimens taken from 195 patients with serologically confirmed scrub typhus in three Thai provinces detected the 56-kDa protein-encoding gene from Orientia tsutsugamushi in ten (5%) patients. Significant genetic diversity was found among the ten amplicons, with nine new genotypes identified that were different from those found previously in Thailand. Phylogenetically, the ten sequences obtained in the present study and sequences from 71 strains characterised previously were distributed into several clusters that included the Karp, Gilliam, Kuroki, Saitama, Kawasaki and Kato clusters. Two of the new genotypes found in the present study clearly belonged to the Karp cluster. However, the other new genotypes formed three different clusters, including one cluster that appeared to be distant from all previously known clusters, and which may therefore be representative of a previously undescribed serotype. Other genotypes formed two other clusters that may also be associated with undescribed serotypes.