NDM-1-producing Klebsiella pneumoniae resistant to colistin in a French community patient without history of foreign travel.

A carbapenem-resistant Klebsiella pneumoniae strain, Kp5196, was responsible for an uncomplicated cystitis in a patient living at home and without history of foreign travel. This isolate produced the metallocarbapenemase NDM-1 and was resistant to all antibiotics except tetracyclines and colistin. The K. pneumoniae strain belonged to sequence type ST15, and bla(NDM-1) was carried by a nontypeable conjugative plasmid. Two months later, a similar ST15 isolate, Kp5241, was present in the patient but was additionally colistin resistant.

Duplication of the chromosomal blaSHV-11 gene in a clinical hypermutable strain of Klebsiella pneumoniae.

In a collection of 110 clinical isolates of Klebsiella pneumoniae, a single strain, Kp593, was found to exhibit a mutator phenotype with a rifampicin mutation frequency 100-fold higher than the modal value for this species. Complementation experiments with the wild-type MutL, one of the main components of the methyl-directed mismatch repair system, allowed the mutator phenotype to be reversed. Sequencing revealed substitution of the conserved residue Lys307 to Arg and site-directed mutagenesis followed by complementation experiments confirmed the critical role of this mutation. The patient infected with Kp593 relapsed a month later and the strain isolated then, Kp869, was identical to Kp593, as verified by PFGE analysis. Phenotypically, Kp869 colonies were more mucoid than those of Kp593, probably due to increased capsule synthesis as shown by electron microscopy. In addition, Kp869 exhibited a 16-fold higher amoxicillin resistance level related to a 36.4 kb tandem duplication encompassing the chromosomal bla(SHV-11) gene, which was unstable in vitro. These data suggest that the mutator phenotype found in Kp593/Kp869 is associated with beneficial mutations conferring a selective advantage, such as increased virulence factor production and antibiotic resistance. The latter was due to resistance gene duplication, an event rarely described in natural isolates. This is the first description of the in vivo occurrence of gene duplication in a mutator background.

CTX-M-producing Escherichia coli in a maternity ward: a likely community importation and evidence of mother-to-neonate transmission.

OBJECTIVES: To investigate the high prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli (4%, 10/250 consecutive isolates) recovered during a 5 month period in the maternity ward of the University Hospital of Bordeaux, France. METHODS: beta-Lactam resistance transfer was analysed by conjugation and transformation. ESBLs were characterized by isoelectric focusing, PCR amplification and sequencing. The relatedness of the strains was examined by PFGE and phylogenetic group determination. Plasmids were characterized by incompatibility group and restriction analysis. RESULTS: Ten ESBL-producing E. coli were isolated from urinary or genital samples of eight mothers and from gastric fluids of two newborns of carrier mothers. The patients were hospitalized in five different units of the maternity ward. Transconjugants, obtained for 7 of the 10 strains, and wild-type strains exhibited various antibiotypes. Different CTX-M enzymes were characterized: CTX-M-1 (n = 4); CTX-M-14 (n = 3); CTX-M-32 (n = 2); and CTX-M-28 (n = 1). The strains recovered from two mothers and their respective babies were identical. All the other strains were epidemiologically unrelated. Furthermore, various plasmids were identified. Environmental samples from the common echographic and sampling rooms did not reveal the presence of ESBL-producing enterobacteria. CONCLUSIONS: The data argue against the occurrence of a nosocomial outbreak and support the hypothesis of an importation of community-acquired ESBL-producing strains into the hospital through colonized/infected patients. At present, not only patients transferred from other hospitals or long-term care facilities are at risk of carrying ESBL-producing enterobacteria on hospital admission, but also community patients.

Molecular and biochemical characterization of SHV-56, a novel inhibitor-resistant beta-lactamase from Klebsiella pneumoniae.

A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla(SHV-56), encoding a new inhibitor-resistant beta-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.

Beta-lactam and aminoglycoside resistance rates and mechanisms among Pseudomonas aeruginosa in French general practice (community and private healthcare centres).

OBJECTIVES: The aim of this study was to assess antibiotic resistance rates and mechanisms of beta-lactam and aminoglycoside resistance among isolates of Pseudomonas aeruginosa isolated in the extra-hospital setting (community and private healthcare centres). PATIENTS AND METHODS: During a 4 month period, 226 non-repetitive strains of P. aeruginosa were collected from patients residing in private healthcare centres (73.5%) or at home (26.5%). Resistance rates were evaluated by MIC determination, and beta-lactam and aminoglycoside resistance was analysed by phenotypic tests, PCR amplification, cloning and sequencing. RESULTS: Among the ticarcillin-resistant strains (38.1%), 33.7% overexpressed their chromosomal cephalosporinase, 27.9% produced acquired penicillinases (21 PSE-1, 2 OXA-21 and 1 TEM-2), 4.7% produced extended-spectrum beta-lactamases (ESBLs) (3 TEM-21 and 1 SHV-2a) and 45.3% possessed a non-enzymatic resistance (NER). Thus, 88.4% had a single mechanism of resistance, whereas 11.6% cumulated several mechanisms. No carbapenemases were detected among the 6.6% imipenem-resistant strains. With regard to aminoglycosides, 23.0% of the strains exhibited an acquired resistance to gentamicin (GEN), tobramycin (TOB), amikacin (AMK) or netilmicin (NET). Enzymatic resistance was more frequent (71.2%) than NER (34.6%). Various aminoglycoside modifying enzymes were associated with overlapping phenotypes: 36.5% strains produced AAC(6')-I with either a serine (GEN-TOB-NET) or a leucine (TOB-NET-AMK) at position 119, or both variants (GEN-TOB-NET-AMK); 21.2% expressed ANT(2'')-I (GEN-TOB), 7.7% AAC(3)-II (GEN-TOB-NET), 5.8% AAC(3)-I (GEN) and 1.9% AAC(6')-II (GEN-TOB-NET-AMK) or AACA7 (TOB-NET-AMK). CONCLUSIONS: Antibiotic resistance rates in P. aeruginosa were globally similar in general practice as in French hospitals. This first analysis of resistance mechanisms showed an unexpectedly high frequency of ESBLs and an unusual distribution of aminoglycoside modifying enzymes.

High Prevalence of Gut Microbiota Colonization with Broad-Spectrum Cephalosporin Resistant Enterobacteriaceae in a Tunisian Intensive Care Unit.

Healthcare-associated infections due to cefotaxime-resistant (CTX-R) Enterobacteriaceae have become a major public health threat, especially in intensive care units (ICUs). Often acquired nosocomially, CTX-R Enterobacteriaceae can be introduced initially by patients at admission. This study aimed to determine the prevalence and genetic characteristics of CTX-R Enterobacteriaceae-intestinal carriage in ICU patients, to evaluate the rate of acquisition of these organisms during hospitalization, and to explore some of the associated risk factors for both carriage and acquisition. Between December 2014 and February 2015, the 63 patients admitted in the ICU of Charles Nicolle hospital were screened for rectal CTX-R Enterobacteriaceae colonization at admission and once weekly thereafter to identify acquisition. CTX-R Enterobacteriaceae fecal carriage rate was 20.63% (13/63) at admission. Among the 50 non-carriers, 35 were resampled during their hospitalization and the acquisition rate was 42.85% (15/35). Overall, 35 CTX-R Enterobacteriaceae isolates were collected from 28 patients (25 Klebsiella pneumoniae, seven Escherichia coli, and three Enterobacter cloacae strains). Seven patients were simultaneously colonized with two CTX-R Enterobacteriaceae isolates. CTX-M-15 was detected in most of the CTX-R Enterobacteriaceae isolates (30/35, 88.23%). Three strains co-produced CMY-4 and 22 strains were carbapenem-resistant and co-produced a carbapenemase [OXA-48 (n = 13) or NDM-1 (n = 6)]. Molecular typing of K. pneumoniae strains, revealed eight Pulsed field gel electrophoresis (PFGE) patterns and four sequence types (ST) [ST101, ST147, ST429, and ST336]. However, E. coli isolates were genetically unrelated and belonged to A (n = 2), B1 (n = 2) and B2 (n = 3) phylogenetic groups and to ST131 (two strains), ST572 (two strains), ST615 (one strain) and ST617 (one strain). Five colonized patients were infected by CTX-R Enterobacteriaceae (four with the same strain identified from their rectal swab and one with a different strain). Whether imported or acquired during the stay in the ICU, colonization by CTX-R Enterobacteriaceae is a major risk factor for the occurrence of serious nosocomial infections. Their systematic screening in fecal carriage is mandatory to prevent the spread of these multidrug resistant bacteria.

High genetic stability of integrons in clinical isolates of Shigella spp. of worldwide origin.

Over a 12-year period, 68 Shigella strains (31 S. sonnei, 30 S. flexneri, 4 S. dysenteriae, and 3 S. boydii strains) were collected in a French University Hospital from the stools of patients who generally had a recent history of travel to various parts of the world (91%), particularly Africa (67%). These strains were often resistant (streptomycin, spectinomycin, trimethoprim, tetracycline, and sulfonamides, 66 to 84%; ampicillin and chloramphenicol, 34 to 38%; nalidixic acid, 4%) and even multiresistant (87%), and they generally carried integrons (81%) of class 1 (21%), class 2 (47%), or both (13%). Class 1 integrons were associated with ampicillin resistance due to the production of an OXA-30 beta-lactamase in S. flexneri and S. dysenteriae. Class 2 integrons were associated with trimethoprim resistance in S. sonnei. Class 1 and class 2 integrons were inserted within transposons Tn21 and Tn7, respectively, themselves located on the bacterial chromosome, except in one strain. Class 1 integrons showed an atypical organization consisting of the insertion sequence IS1 at the 3' end instead of the typical 3' conserved segment and two blaOXA-30 and aadA1 gene cassettes, despite the absence of epidemiological relationships between the strains, and an apparently functional integrase. Class 2 integrons showed the same albeit classical organization with the three dfrA1, sat, and aadA1 gene cassettes. Occasionally, the 3' end was deleted and the aadA1 gene cassette was unexpressed. Thus, integrons contributed only in part to the multidrug resistance of the Shigella strains. The highly conserved organization of integrons might be related to their location within mobile genetic superstructures.

Extended-spectrum-beta-lactamase-producing Enterobacteriaceae strains in various types of private health care centers.

During a 2004 survey, 49 extended-spectrum-beta-lactamase-producing enterobacteria were collected in 20 French private health care centers and one local hospital. They included 12 CTX-M-producing Escherichia coli strains (1.8% versus 0.3% in a 1999 survey). Most of them belonged to the same clone and contained a bla(CTX-M-15) gene on similar conjugative plasmids.

Prolonged outbreak of infection due to TEM-21-producing strains of Pseudomonas aeruginosa and enterobacteria in a nursing home.

Over a 6-year period, 24 extended-spectrum beta-lactamase (ESBL)-producing isolates of Pseudomonas aeruginosa were collected from 18 patients living in a nursing home. These isolates had a delayed development of a red pigment and exhibited a similar antibiotype (resistance to all beta-lactams except for imipenem and to gentamicin, tobramycin, netilmicin, ciprofloxacin, and rifampin) associated with the production of the TEM-21 beta-lactamase and a type II 3'-N-aminoglycoside acetyltransferase [AAC(3)-II] enzyme. Surprisingly, serotyping showed that these isolates belonged to four successive serotypes (P2, P16, P1, and PME), although molecular typing by PCR methods and pulsed-field gel electrophoresis yielded identical or similar profiles. Moreover, in all isolates the bla(TEM-21) gene was part of a chromosomally located Tn801 transposon truncated by an IS6100 element inserted within the resolvase gene, and the aac(3)-II gene was adjacent to this structure. During the same period, 17 ESBL-producing isolates of enterobacteria were also collected from 10 of these patients. These isolates harbored a similar large plasmid that contained the bla(TEM-21) and the aac(3)-II genes and that conferred additional resistance to sulfonamides and chloramphenicol, as well as to kanamycin, tobramycin, netilmicin, and amikacin, conveyed by an AAC(6')-I enzyme. The bla(TEM-21) gene was part of the Tn801 transposon disrupted by IS4321. Thus, a single clone of P. aeruginosa that had undergone a progressive genetic drift associated with a change in serotype appeared to be responsible for an outbreak of nosocomial infections in a nursing home. This strain has probably acquired the bla(TEM-21)-encoding plasmid that was epidemic among the enterobacteria at the institution, followed by chromosomal integration and genomic reorganization.

SHV-49, a novel inhibitor-resistant beta-lactamase in a clinical isolate of Klebsiella pneumoniae.

A clinical strain of Klebsiella pneumoniae carried the bla(SHV-49) gene, encoding a novel inhibitor-resistant beta-lactamase of pI 7.6, derived from SHV-1 by the single substitution M69I. It also harbored a gene differing from bla(SHV-11) by four silent mutations and coding for a penicillinase. Both genes were chromosome located and might represent either a species-specific gene or an acquired resistance gene.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in community and private health care centers.

In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum beta-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.