Highlighting extended-spectrum beta-lactamase-producing Klebsiella pneumoniae outbreak by routine genomic typing.

Whole genome sequencing has become the gold standard for any microbiological investigations. Taking the opportunity to doing it prospectively and routinely allowed to detect undeclared outbreaks. Thanks to that, we investigated and ended a rare epidemic extended-spectrum beta-lactamase-producing Klebsiella pneumoniae ST584 strain on two intensive care units over a 4-month period.

Evaluation of Flow Cytometry for Cell Count and Detection of Bacteria in Biological Fluids.

The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The gold standard method for cell count in biological fluids is the manual method using counting chambers. The microbiological routine procedures consist of Direct Gram staining and culture on solid or liquid media. We evaluate the analytical performance of SYSMEX UF4000 (Sysmex, Kobe, Japan) and Sysmex XN10 (Sysmex, Kobe, Japan) in comparison with cytological and microbiological routine procedures. A total of 526 biological fluid samples were included in this study (42 ascitic, 31 pleural, 31 peritoneal, 125 cerebrospinal, 281 synovial, and 16 peritoneal dialysis fluids). All samples were analyzed by flow cytometry and subsequently processed following cytological and/or microbiological routine procedures. With regard to cell counts, UF4000 (Sysmex, Kobe, Japan) showed a performance that was at least equivalent to those of the reference methods and superior to those of XN10 (Sysmex, Kobe, Japan). Moreover, the bacterial count obtained with UF4000 (Sysmex, Kobe, Japan) was significantly higher among culture or Direct Gram stain positive samples. We established three optimal cutoff points to predict Direct Gram stain positive samples for peritoneal (465.0 bacteria/µL), synovial (1200.0 bacteria/µL), and cerebrospinal fluids (17.2 bacteria/µL) with maximum sensitivity and negative predictive values. Cell count and detection of bacteria by flow cytometry could be used upstream cytological and microbiological routine procedures to improve and accelerate the diagnosis of infection of biological fluid samples. IMPORTANCE The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The possibility of carrying out cytological and microbiological analyses of biological fluid samples on the same automated machine would simplify the sample circuit (addressing the sample in a single laboratory, 24/7). It would also minimize the quantity of sample required. The performance of cytological and microbiological analysis by the flow cytometer UF 4000 (Sysmex, Kobe, Japan) has never been evaluated yet. This study has shown that bacterial count by flow cytometry using UF4000 (Sysmex, Kobe, Japan) could be used upstream of microbiological routine procedures to improve and to accelerate the diagnosis of infection of biological fluid samples.

Investigation of Serratia marcescens surgical site infection outbreak associated with peroperative ultrasonography probe.

Early postoperative infections due to Serratia marcescens have been reported by both clinicians and microbiologists in our teaching hospital. Here, we present an interlinked clinical, epidemiological, environmental and genomic investigation of this outbreak due to a T-shaped intraoperative probe contaminated by S. marcescens used during peroperative ultrasonography in laparoscopic liver resection.

Outbreak of Salmonella Newport associated with internationally distributed raw goats' milk cheese, France, 2018.

Raw milk cheeses are commonly consumed in France and are also a common source of foodborne outbreaks (FBOs). Both an FBO surveillance system and a laboratory-based surveillance system aim to detect Salmonella outbreaks. In early August 2018, five familial FBOs due to Salmonella spp. were reported to a regional health authority. Investigation identified common exposure to a raw goats' milk cheese, from which Salmonella spp. were also isolated, leading to an international product recall. Three weeks later, on 22 August, a national increase in Salmonella Newport ST118 was detected through laboratory surveillance. Concomitantly isolates from the earlier familial clusters were confirmed as S. Newport ST118. Interviews with a selection of the laboratory-identified cases revealed exposure to the same cheese, including exposure to batches not included in the previous recall, leading to an expansion of the recall. The outbreak affected 153 cases, including six cases in Scotland. S. Newport was detected in the cheese and in the milk of one of the producer's goats. The difference in the two alerts generated by this outbreak highlight the timeliness of the FBO system and the precision of the laboratory-based surveillance system. It is also a reminder of the risks associated with raw milk cheeses.

Hemolytic uremic syndrome due to Shiga toxin-producing Escherichia coli infection.

The leading cause of hemolytic uremic syndrome (HUS) in children is Shiga toxin-producing Escherichia coli (STEC) infection, which has a major outbreak potential. Since the early 2010s, STEC epidemiology is characterized by a decline of the historically predominant O157 serogroup and the emergence of non-O157 STEC, especially O26 and O80 in France. STEC contamination occurs through the ingestion of contaminated food or water, person-to-person transmission, or contact with ruminants or their contaminated environment. The main symptom is diarrhea, which is bloody in about 60% of patients and occurs after a median incubation period of three days. Shiga toxins released by STEC induce a cascade of thrombogenic and inflammatory changes of microvascular endothelial cells. HUS is observed in 5-15% of STEC infection cases, defined by the triad of mechanical hemolytic anemia, thrombocytopenia, and acute renal injury. The diagnosis of STEC infection relies on biological screening for Shiga toxins and STEC in stools and serology. Treatment of STEC-HUS is mainly symptomatic, as no specific drug has proved effective. The effect of antibiotics in STEC infection and STEC-HUS remains debated; however, some bacteriostatic antibiotics might have a beneficial effect. Proofs of evidence of a benefit from complement blockade therapy in STEC-HUS are also lacking. Clinical and bacteriological STEC-HUS surveillance needs to be continued. Ongoing prospective studies will document the role of bacteriostatic antibiotics in STEC infection and STEC-HUS, and of complement blockade therapy in STEC-HUS.

The French human Salmonella surveillance system: evaluation of timeliness of laboratory reporting and factors associated with delays, 2007 to 2011.

Given the regular occurrence of salmonellosis outbreaks in France, evaluating the timeliness of laboratory reporting is critical for maintaining an effective surveillance system. Laboratory-confirmed human cases of Salmonella infection from whom strains were isolated from 2007 to 2011 in France (n=38,413) were extracted from the surveillance database. Three delay intervals were defined: transport delay (strain isolation, transport from primary laboratory to national reference laboratory), analysis delay (serotyping, reporting) and total reporting delay. We calculated the median delay in days and generated the cumulative delay distribution for each interval. Variables were tested for an association with reporting delay using a multivariable generalised linear model. The median transport and analysis delays were 7 and 6 days respectively (interquartile range (IQR: 6-10 and 4-9 respectively), with a median total reporting delay of 14 days (IQR: 11-19). Timeliness was influenced by various external factors: decreasing serotype frequency, geographical zone of primary laboratory and strain isolation on Sundays were the variables most strongly associated with increased length of delay. The effect of season and day of the week of isolation was highly variable over the study period. Several areas for interventions to shorten delays are identified and discussed for both transport and analysis delays.

Prevalence and characterization of extended-spectrum ß-lactamase-producing clinical Salmonella enterica isolates in Dakar, Senegal, from 1999 to 2009.

A total of 1623 clinical isolates of Salmonella belonging to 229 serotypes were received by the Senegalese Reference Center for Enterobacteria from January 1999 to December 2009. The most common serotypes were Enteritidis (19% of the isolates), Typhi (8%), Typhimurium (7%) and Kentucky (4%). A significant increase in the prevalence of resistance to amoxicillin (0.9% in 1999 to 11.1% in 2009) and nalidixic acid (0.9% in 1999 to 26.7% in 2009) was observed in non-typhoidal Salmonella serotypes. For critically important antibiotics, notably ciprofloxacin and extended-spectrum cephalosporins (ESCs), the rates of resistance were low: 0.3% and 0.5%, respectively. Seven ESC-resistant Salmonella strains and three additional ESC-resistant strains from Senegal (1990) and Mali (2007) were studied to identify the genetic basis of their antibiotic resistance. All ESC-resistant strains produced an extended-spectrum ß-lactamase (ESBL). These were CTX-M-15 (n = 6; 2000-2008), SHV-12 (n = 3; 2000-2001) and SHV-2 (n = 1; 1990). A large IncHI2 ST1 pK29-like plasmid was found in six strains (three producing SHV-12 and three CTX-M-15), whereas IncN and IncF plasmids were found in three strains and one strain, respectively. The association of plasmid-mediated quinolone resistance (PMQR) genes qnrB1 and aac(6')-Ib-cr was found in four ESBL-producing strains, leading to decreased susceptibility and even full resistance to ciprofloxacin (MIC range 0.75-2 mg/L) despite the absence of mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC and parE. This association of ESBL and multiple PMQR mechanisms within the same strains is therefore a serious concern as it hampers the use of both ESCs and fluoroquinolones for severe Salmonella infections.

Unusual increase in reported cases of paratyphoid A fever among travellers returning from Cambodia, January to September 2013.

From January to September 2013, a marked increase in notifications of Salmonella Paratyphi A infections among travellers returning from Cambodia occurred in France. An investigation revealed 35 cases without a common source: 21 in France, five in Germany, three in the Netherlands, one in Norway, one in the United Kingdom, four in New-Zealand. Data suggest an ongoing event that should trigger further investigation. Travellers to Cambodia should observe preventive measures including good personal hygiene and food handling practices.

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

Epidemiological and microbiological investigation of a large outbreak of monophasic Salmonella Typhimurium 4,5,12:i:- in schools associated with imported beef in Poitiers, France, October 2010.

An outbreak due to the emerging monophasic Salmonella Typhimurium 4,5,12:i:- occurred in four schools in Poitiers in October 2010. Food trace-back investigation led to the identification of beef burgers as the cause of the outbreak and their subsequent withdrawal. The Institute for Public Health Surveillance conducted a retrospective epidemiological investigation to assess the extent of the outbreak and describe cases. Self-administered questionnaires were completed by students and personnel attending each of the four schools affected. Clinical cases were defined as anyone having eaten at the school when the beef burgers were served and reporting diarrhoea or fever with at least one digestive symptom (nausea, vomiting or abdominal pain), within five days after the incriminated school meal or with unknown date of onset within a 15-day period after the incriminated school meal. Of 1,559 persons exposed, 554 clinical cases were identified corresponding to an overall attack rate of 35.5%. Of 554 clinical cases, a total of 286 (53%) sought medical care and 31 (6%) were hospitalised for more than 24 hours. This multi-school outbreak is one of the biggest food-borne outbreaks of monophasic Salmonella Typhimurium 4,5,12:i:- described in France. Prompt notification of cases and rapid identification and withdrawal of the incriminated batch of beef burgers was crucial to limit the extension of this outbreak.

Nationwide outbreak of Salmonella enterica serotype 4,[5],12:i:- infection associated with consumption of dried pork sausage, France, November to December 2011.

An outbreak of the monophasic variant of Salmonella enterica serotype 4,[5],12:i:- occurred in November and December 2011 in France. Epidemiological investigation and food investigation with the help of supermarket loyalty cards suggested dried pork sausage from one producer as the most likely source of the outbreak. Despite the absence of positive food samples, control measures including withdrawal and recall were implemented.

Invasive serotype 1 Streptococcus pneumoniae outbreaks in the South Pacific from 2000 to 2007.

In New Caledonia, Wallis and Futuna, and French Polynesia, an active surveillance system was established to monitor pneumococcal serotype prevalence between 2000 and 2007. The most prevalent serotype was serotype 1, which belonged to the major clonal complex sequence type 306 (ST306) and was responsible for invasive pneumococcal disease outbreaks.

Nationwide outbreak of Salmonella enterica serotype 4,12:i:- infections in France, linked to dried pork sausage, March-May 2010.

In May 2010, a nationwide excess of infections with the specific monophasic variant Salmonella enterica serotype 4,12:i:- was investigated in France. Subtyping with multilocus variable number of tandem repeats analysis revealed a distinct epidemic strain within this excess. Epidemiological investigations identified a dried pork sausage sold by a particular chain of supermarkets as the likely vehicle of transmission. The suspected batches have been withdrawn and recalled.

Clinical and microbial characteristics of invasive Streptococcus pyogenes disease in New Caledonia, a region in Oceania with a high incidence of acute rheumatic fever.

New Caledonia is an archipelago in the South Pacific with a high prevalence of acute rheumatic fever and rheumatic heart disease. Conducted in 2006, this study aimed at characterizing clinical manifestations and microbial features of isolates obtained from invasive Streptococcus pyogenes disease. Clinical and demographic data were collected prospectively. Isolates were biotyped, T typed, emm sequenced, and tested for antibiotic susceptibility. Detection of the speA, speB, speC, and ssa genes was also carried out. The estimated annual incidence of invasive S. pyogenes disease in 2006 was high at 38 cases/100,000 inhabitants in New Caledonia. Invasive isolates were obtained from 90 patients with necrotizing fasciitis (41 cases), bacteremia with no identified focus (12 cases), myositis (10 cases), septic arthritis (9 cases), erysipelas (8 cases), postpartum infection (4 cases), myelitis and osteomyelitis (3 cases), severe pneumonia (2 cases), and endocarditis (1 case). The most frequent associated comorbidities were skin lesions (71%) and obesity (29%). Thirty-one different emm types were identified, and the following six accounted for 54% of the isolates: emm15 (15.5%), emm92 (12.2%), emm106 (8.9%), emm74 (6.7%), emm89 (5.6%), and emm109 (5.6%). The speA, speC, and ssa genes were expressed at different frequencies in the various emm types. The first epidemiological study of invasive S. pyogenes disease in New Caledonia highlights that emm type distribution is particular and should be taken into account in the development of an appropriate vaccine. These findings support the prevention of pyoderma and other cutaneous lesions in order to limit the development of both invasive disease and poststreptococcal sequelae in the South Pacific.

Excess of infections due to a multi-drug sensitive Salmonella enterica serotype Typhimurium in France in June 2008.

An unusually high number of cases of Salmonella Typhimurium was reported in France in June 2008. In the course of epidemiological investigations 112 cases were ascertained, of whom 75 were interviewed. Subtyping by PFGE and MLVA identified a strain named "majority profile". Subtyping results were available for 45 interviewed cases, 30 of whom (majority below 15 years of age) were found to be infected with the majority profile strain. Evidence suggested the occurrence of an outbreak due to a monoclonal S. Typhimurium strain with the single PFGE profile XTYM-50. Cases with identical PFGE profile were also detected in Switzerland but no link with outbreaks occurring in the same period in Denmark and in the Netherlands was found. Contamination of a product distributed nationally was suggested as the cause of the outbreak but investigations did not reveal any specific food source.

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in New Caledonia.

Carbapenem-resistant Acinetobacter baumannii (CR-Ab) ranked third, with a frequency of 24.8%, among 202 strains of multidrug-resistant bacteria isolated from clinical samples in the main hospital of New Caledonia in 2004. All CR-Ab isolates were analysed by isoelectric focusing, conjugation, pulsed-field gel electrophoresis and PCR for the presence of carbapenemase genes. Fifty CR-Ab isolates produced carbapenemase OXA-23. The isolates belonged to a single clone presenting several subtypes, suggesting an endemic situation. This study further illustrates the widespread prevalence of carbapenemase OXA-23-producing CR-Ab isolates in the South Pacific.

Real-time PCR detection of gyrA and parC mutations in Streptococcus pneumoniae.

Fluoroquinolone resistance in Streptococcus pneumoniae mainly involves stepwise mutations predominantly in the parC and gyrA genes. We have developed a single-run real-time PCR assay for detection of the four most common mutations in the quinolone resistance-determining regions of these genes. This assay provides a useful tool for both clinical and epidemiological use.

High prevalence of hepatitis C virus infection and predominance of genotype 4 in rural Gabon.

Hepatitis C (HCV) molecular epidemiology is documented poorly in central African countries. In response to this, a population-based study of 319 consenting adults resident in a remote village of Gabon was undertaken (mean age: 38 years; age range: 13-85+; sex ratio: 0.74). Screening for anti-HCV antibodies was performed using ELISA and recombinant immunoblot assay. Seropositive samples were assessed further with viral load and genotyping techniques. Sixty-six (20.7%) individuals were HCV seropositive. Viral loads ranged from 600 to 24.9 million IU/ml (median: 372,500). Seroprevalence and viral loads increased significantly with age (P < 10(-5) and P < 0.003, respectively). HCV sequences of the 5'UTR genome region were obtained from 60 (90.9%) samples and NS5B region sequences were obtained from 22 (36.6%) samples. All strains belonged to subtypes of genotype 4: 4e (72.7%), 4c (13.6%), 4p (4.5%), 4r (4.5%) and one unclassified genotype 4 strain. Evolutionary analysis of the subtype 4e sequences indicates a period of raised transmission during the early twentieth century.