Evidence of Sexual Transmission of Extended-Spectrum ß-Lactamase-Producing Enterobacterales: A Cross-sectional and Prospective Study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) represent a major threat to public health. Little is known on their potential for sexual transmission. METHODS: We recruited individuals at a sexually transmitted infection and human immunodeficiency virus (HIV) outpatient clinic in Paris, France, in whom we evaluated the prevalence of ESBL-E intestinal carriage and, among those testing positive, the proportion with clearance 6 months thereafter. We compared carriage prevalence between groups using logistic regression adjusted for age, geographic origin, travel outside Europe, and antibiotic use in the past 6 months. RESULTS: A total of 2157 individuals participated, of whom 226 (10.5%) were ESBL-E carriers. The proportions of ESBL-E carriers varied across sexual groups and were as follows: HIV-negative men who have sex with men (MSM) and who were on preexposure prophylaxis (PrEP), 16.3% (41 of 251); HIV-negative MSM not on PrEP, 9.7% (47 of 487); HIV-positive MSM, 12.2% (61 of 500); HIV-negative men who have sex exclusively with women, 10.0% (44 of 439); and HIV-negative women who have sex with men, 6.9% (n = 33 of 480). After adjustment, ESBL-E prevalence was significantly higher in HIV-negative MSM on PrEP (P < .001) and HIV-positive MSM (P = .01) than in women who have sex with men. A higher number of sexual partners in the past 6 months was associated with ESBL-E carriage after adjustment (P = .004). Escherichia coli sequence type 14 and blaSHV-12-producing ESBL-E were observed only in MSM. Of 102 individuals with ESBL-E returning for testing, 26 (25%) had carriage at 6 months. CONCLUSION: ESBL-E carriage is more frequent in MSM undergoing PrEP or living with HIV and with increasing number of sexual partners. More research is warranted to understand the consequences of ESBL-E carriage in these populations and how transmission can be reduced.

Spread of NDM-5 and OXA-181 Carbapenemase-Producing Escherichia coli in Chad.

We detected for the first time blaNDM-5 and blaOXA-181 in Escherichia coli isolates from hospitalized patients and healthy volunteers in Chad. These resistance genes were located on IncX3 and IncF plasmids. Despite the large diversity of E. coli clones, the identified resistant intestinal isolates belonged mainly to the same sequence type.

Biofilm formation by ESBL-producing strains of Escherichia coli and Klebsiella pneumoniae.

OBJECTIVES: Biofilm production in extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae provides a favourable environment for the exchange of antibiotic-resistance genes and could facilitate widespread dissemination. We aimed to assess biofilm development in ESBL-producing E. coli and K. pneumoniae isolates and determine how development relates to microbiological characteristics and clinical outcomes. METHODS: 147 ESBL-producing E. coli and 82 K. pneumoniae were genetically characterized. Biofilm formation was measured at 1.5, 4, 6, and 24 h during culture in blood heart infusion using a microbead immobilization assay (BioFilm Ring test®). Results were given as biofilm formation index (BFI) with lower values indicating increased presence of biofilm (range = 0-21). RESULTS: In total, 57.1% of strains were strong producers of biofilm (BFI < 2), whereas 13.4% lacked biofilm production (BFI > 18). Standard biofilm production (BFI < 7) was common in E. coli isolates (61.9%). For E. coli, biofilm production was less frequently observed in ST131 clones (p = 0.03) but more frequently in strains harbouring toxin (p = 0.008) or adhesin (p = 0.008) virulence factor genes. Despite almost all K. pneumoniae having standard biofilm production (90.2%), there was a 2.4-times higher odds of observing biofilm in ST29/147/323 versus other ST-types (p = 0.13). Patients with standard biofilm producing isolates were not at increased risk of transfer to intensive-care (odds-ratio=2.80, 95%CI=0.59-13.21) or death within 12-months (odds-ratio=1.61, 95%CI=0.75-3.43). CONCLUSION: In these ESBL-producing strains, biofilm production is linked to certain virulence factors in E. coli and is common in K. pneumoniae. Further exploration of whether biofilm production increases dissemination and risk of severe clinical outcomes is needed in larger collections of isolates.

Chlamydia trachomatis screening in urine among asymptomatic men attending an STI clinic in Paris: a cross-sectional study.

BACKGROUND: The incidence of Chlamydia trachomatis (Ct) urethritis has been increasing for the past 10 years. There is little data regarding the screening of Ct infection in asymptomatic men in France, despite the national recommendation to screen at-risk asymptomatic men under 30 attending Sexually Transmitted Infections (STI) clinics. Recent data from the French surveillance network Rénachla show indeed that systematic screening is still focused on women. The objective of our study was to determine the prevalence and risk factors for Ct infection in asymptomatic men under 30 attending an STI clinic located in Paris, France. METHODS: We performed a cross-sectional study between April 4, and December 31, 2016 in the database of the software DIAMM Client V8 used in our STI clinic. We extracted the demographic characteristics, sexual behavior and result of STI screening of all asymptomatic men who had consulted and given their consent for the use of their personal data. Those data were collected in usual care through a standardized questionnaire filled in during an appointment with a trained physician. STI screening was performed using PCR kit CT/NG Abbott Realtime® on first void urines. For MSM, a rectal swab was also collected. Risk factors for Ct infection were analyzed by univariate and multivariate modeling using STATA software 8.2. RESULTS: Among 872 men who had attended the clinic, 647 were included and 37 (5.7, 95% CI 4.2 to 7.8) were positive for Ct in urine. In univariate analysis, men who had unprotected sex in the last 6 weeks (OR 2.40 (95%CI 1.16 to 4.94), p = 0.02), and those who had an infected partner (OR 7.6 (95%CI 3.03 to 20), p = 0.0001) were more likely to be infected. In the multivariate analysis having an infected partner was the only risk factor (OR 11.1(95% CI 3.7 to 33.3), p = 0.0001) that remained significant. CONCLUSION: Prevalence of Ct infection is high among asymptomatic men of 30 years or less attending our urban STI clinic especially among those with an infected partner. The Ct screening among this population associated with partner notification, as recommended by the French national guidelines, should be more widely implemented.

Within-a-Day Detection and Rapid Characterization of Carbapenemase by Use of a New Carbapenem Inactivation Method-Based Test, CIMplus.

The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major threat to public health. Rapid and accurate detection of CPE is essential for initiating appropriate antimicrobial treatment and establishing infection control measures. The carbapenem inactivation method (CIM), which has good sensitivity and specificity but a detection time of 20 h, was recently described. In this study, we evaluated the performances of a new version, the CIMplus test, which allows detection of carbapenemases in 8 h and characterization of carbapenemase classes, according to the Ambler classification, in 20 h. A panel of 110 carbapenem-resistant Enterobacteriaceae strains, including 92 CPE strains (with NDM, VIM, IMP, KPC, GES, OXA-48, and OXA-48-like enzymes), was used to evaluate test performance. Carbapenemase activity was detected at 8 h and 20 h. Characterization of carbapenemase classes, using specific inhibitors, was possible in 20 h. The CIMplus test had sensitivities of 95.7% and 97.8% at 8 h and 20 h, respectively, and a specificity of 94.4%, independent of the culture duration. Using a decision algorithm, this test was successful in identifying the carbapenemase class for 98.9% of tested CPE isolates (87/88 isolates). In total, the characterization was correct for 100%, 96.9%, and 100% of Ambler class A, B, and D isolates, respectively. Therefore, this test allows detection of carbapenemase activity in 8 h and characterization of carbapenemase classes, according to the Ambler classification, in 20 h. The CIMplus test represents a simple, affordable, easy-to-read, and accurate tool that can be used without any specific equipment.

Emergence of Plasmid-Mediated Fosfomycin-Resistance Genes among Escherichia coli Isolates, France.

FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum ß-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.

Diversity and functionality of plasmid-borne VagCD toxin-antitoxin systems of Klebsiella pneumoniae.

Objectives: To explore the VagCD toxin-antitoxin (TA) systems encoded on plasmids in multiresistant Klebsiella pneumoniae strains. Methods: Previously sequenced K. pneumoniae plasmids were used for in silico analysis and a collection of 63 resistant K. pneumoniae strains was used for epidemiological study. Functional analysis was done after separate cloning of the toxin gene under the control of the arabinose-inducible promoter of pBAD43 and of the antitoxin gene under the control of the constitutive promoter of pUC19. Results: In silico , two types of VagCD systems, VagCD1 and VagCD2, encoded on K. pneumoniae plasmids could be distinguished, 15% carrying one of these TA systems. Moreover, in a collection of antibiotic-resistant K. pneumoniae strains including ESBL or carbapenemase producers, 17.5% of isolates were found to harbour a VagCD TA system. VagCD1 and VagCD2 were proved functional TA systems, with VagD the toxin and VagC its antitoxin, not only in K. pneumoniae but also in Escherichia coli and other Enterobacteriaceae. Toxin expression was found to induce a significant decrease in a bacterial population resulting from both bactericidal and bacteriostatic effects. Conclusions: The vagCD genes of K. pneumoniae encode a functional broad-spectrum TA system and are conserved on the large multiple antibiotic resistance-conferring plasmids in this species.

Cooccurrence of Multiple AmpC ß-Lactamases in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in Tunisia.

Over a period of 40 months, plasmid-mediated AmpC ß-lactamases were detected in Tunis, Tunisia, in 78 isolates (0.59%) of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. In 67 isolates, only one ampC gene was detected, i.e., blaCMY-2-type (n = 33), blaACC (n = 23), blaDHA (n = 6) or blaEBC (n = 5). Multiple ampC genes were detected in 11 isolates, with the following distribution: blaMOX-2, blaFOX-3, and blaCMY-4/16 (n = 6), blaFOX-3 and blaMOX-2 (n = 3), and blaCMY-4 and blaMOX-2 (n = 2). A great variety of plasmids carrying these genes was found, independently of the species and the bla gene. If the genetic context of blaCMY-2-type is variable, that of blaMOX-2, reported in part previously, is unique and that of blaFOX-3 is unique and new.

High Prevalence of SXT/R391-Related Integrative and Conjugative Elements Carrying blaCMY-2 in Proteus mirabilis Isolates from Gulls in the South of France.

The genetic structures involved in the dissemination of blaCMY-2 carried by Proteus mirabilis isolates recovered from different gull species in the South of France were characterized and compared to clinical isolates. blaCMY-2 was identified in P. mirabilis isolates from 27/93 yellow-legged gulls and from 37/65 slender-billed gulls. It was carried by a conjugative SXT/R391-like integrative and conjugative element (ICE) in all avian strains and in 3/7 human strains. Two clinical isolates had the same genetic background as six avian isolates.

Did L Strains Responsible for Lymphogranuloma Venereum Proctitis Spread Among People With Genital Chlamydia trachomatis Infection in France in 2013?

We retrospectively analyzed 1802 nonrectal Chlamydia trachomatis-positive specimens to determine if the L strains responsible for rectal Lymphogranuloma venereum in men who have sex with men could spread to the heterosexual population. No evidence for Lymphogranuloma venereum transmission among heterosexuals in France was observed in 2013. L2b strains seem to be restricted to the men who have sex with men population.

Extended-spectrum beta-lactamase--producing enterobacteriaceae in the intensive care unit: acquisition does not mean cross-transmission.

BACKGROUND: In intensive care unit (ICU), infection and colonization by resistant Gram-negative bacteria increase costs, length of stay and mortality. Extended-spectrum beta-lactamase--producing Enterobacteriaceae (ESBL-E) is a group of pathogens increasingly encountered in ICU setting. Conditions that promote ESBL-E acquisition are not completely understood. The increasing incidence of infections related to ESBL-E and the unsolved issues related to ESBL-E cross-transmission, prompted us to assess the rates of referred and acquired cases of ESBL-E in ICU and to assess patient-to-patient cross-transmission of ESBL-E using a multimodal microbiological analysis. METHODS: During a 5-month period, all patients admitted to a medical ICU were tested for ESBL-E carriage. A rectal swab was performed at admission and then twice a week until discharge or death. ESBL-E strains were analyzed according to antibiotic susceptibility pattern, rep-PCR (repetitive-element Polymerase chain reaction) chromosomal analysis, and plasmid PCR (Polymerase chain reaction) analysis of ESBL genes. Patient-to-patient transmission was deemed likely when 2 identical strains were found in 2 patients hospitalized simultaneously in the ICU. RESULTS: Among the 309 patients assessed for ESBL-E carriage on admission, 25 were found to carry ESBL-E (importation rate: 8%). During follow-up, acquisition was observed among 19 of them (acquisition rate: 6.5%). Using the multimodal microbiological approach, we found only one case of likely patient-to-patient ESBL-E transmission. CONCLUSIONS: In unselected ICU patients, we found rather low rates of ESBL-E referred and acquired cases. Only 5% of acquisitions appeared to be related to patient-to-patient transmission. These data highlight the importance of jointly analyzing phenotypic profile and molecular data to discriminate strains of ESBL-E.

High prevalence of extended-spectrum ß-lactamase producing enterobacteriaceae among clinical isolates in Burkina Faso.

BACKGROUND: Nothing is known about the epidemiology and resistance mechanisms of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) in Burkina Faso. The objective of this study was to determine ESBL-PE prevalence and to characterize ESBL genes in Burkina Faso. METHODS: During 2 months (June-July 2014), 1602 clinical samples were sent for bacteriologic investigations to the microbiology laboratories of the tree main hospitals of Burkina Faso. Isolates were identified by mass spectrometry using a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) BioTyper. Antibiotic susceptibility was tested using the disk diffusion method on Müller-Hinton agar. The different ESBL genes in potential ESBL-producing isolates were detected by PCR and double stranded DNA sequencing. Escherichia coli phylogenetic groups were determined using a PCR-based method. RESULTS: ESBL-PE frequency was 58 % (179 strains among the 308 Enterobacteriaceae isolates identified in the collected samples; 45 % in outpatients and 70 % in hospitalized patients). The CTX-M-1 group was dominant (94 %, CTX-M-15 enzyme), followed by the CTX-M-9 group (4 %). ESBL producers were more often found in E. coli (67.5 %) and Klebsiella pneumoniae (26 %) isolates. E. coli isolates (n = 202; 60 % of all Enterobacteriaceae samples) were distributed in eight phylogenetic groups (A = 49, B1 = 15, B2 = 43, C = 22, Clade I = 7, D = 37, F = 13 and 16 unknown); 22 strains belonged to the sequence type ST131. No association between a specific strain and ESBL production was detected. CONCLUSIONS: This report shows the alarming spread of ESBL genes in Burkina Faso. Public health efforts should focus on education (population and healthcare professionals), surveillance and promotion of correct and restricted antibiotic use to limit their dissemination.

Non-O1/Non-O139 Vibrio cholerae Avian Isolate from France Cocarrying the bla(VIM-1) and bla(VIM-4) Genes.

We describe here a non-O1/non-O139 Vibrio cholerae isolate producing both VIM-1 and VIM-4 carbapenemases. It was isolated from a yellow-legged gull in southern France. The blaVIM genes were part of a class 1 integron structure located in an IncA/C plasmid. This study emphasizes the presence of carbapenemase genes in wildlife microbiota.

Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups.

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.

Complete nucleotide sequence of two multidrug-resistant IncR plasmids from Klebsiella pneumoniae.

We report here the complete nucleotide sequence of two IncR replicons encoding multidrug resistance determinants, including ß-lactam (blaDHA-1, blaSHV-12), aminoglycoside (aphA1, strA, strB), and fluoroquinolone (qnrB4, aac6'-1b-cr) resistance genes. The plasmids have backbones that are similar to each other, including the replication and stability systems, and contain a wide variety of transposable elements carrying known antibiotic resistance genes. This study confirms the increasing clinical importance of IncR replicons as resistance gene carriers.

The ß-Lacta test for direct detection of extended-spectrum-ß-lactamase-producing Enterobacteriaceae in urine.

With the ß-Lacta test, production of extended-spectrum ß-lactamases (ESBLs) was assayed in 200 urine samples showing Gram-negative bacilli during direct microscopic examination. While 168 samples tested negative, all samples yielding ESBL-producing Enterobacteriaceae after culture gave positive (n = 30) or uninterpretable (n = 2) results. The sensitivity and specificity of ESBL detection were 94% and 100%, respectively.

Multiplex PCR for detection of seven virulence factors and K1/K2 capsular serotypes of Klebsiella pneumoniae.

A single multiplex PCR assay targeting seven virulence factors and the wzi gene specific for the K1 and K2 capsular serotypes of Klebsiella pneumoniae was developed and tested on 65 clinical isolates, which included 45 isolates responsible for community-acquired severe human infections. The assay is useful for the surveillance of emerging highly virulent strains.

Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae.

Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called "plasmid relaxase gene typing" (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice.

Complete nucleotide sequence of the first KPC-2- and SHV-12-encoding IncX plasmid, pKpS90, from Klebsiella pneumoniae.

We report the complete nucleotide sequence of the pKpS90 plasmid, carrying the bla(KPC-2) and bla(SHV-12) genes. This plasmid was isolated from a sequence type 258 (ST258) Klebsiella pneumoniae strain responsible for an outbreak in a French university hospital in 2009. pKpS90 is a 53,286-bp plasmid that belongs to the IncX incompatibility group. pKpS90 consists of a backbone from IncX plasmids, in which the KPC-2-encoding Tn4401 transposon and a bla(SHV-12)-encoding region have been inserted.