First Report of KPC-2 and KPC-3-Producing Enterobacteriaceae in Wild Birds in Africa.

The increased incidence of antibiotic-resistant Enterobacteriaceae is a public health problem worldwide. The aim of this study was to analyze the potential role of wild birds, given their capacity of migrating over long distances, in the spreading of carbapenemase, extended-spectrum ß-lactamase (ESBL), and acquired-AmpC beta-lactamase-producing Enterobacteriaceae in the environment. Fecal and pellet samples were recovered from 150 wild birds in seven Tunisian regions and were inoculated in MacConkey-agar plates for Enterobacteriaceae recovery (one isolate/animal). Ninety-nine isolates were obtained and acquired resistance mechanisms were characterized in the five detected imipenem-resistant and/or cefotaxime-resistant isolates, by PCR and sequencing. The following ESBL, carbapenemase, and acquired-AmpC beta-lactamase genes were detected: blaCTX-M-15 (two Escherichia fergusonii and one Klebsiella oxytoca isolates), blaKPC-2 (one K. oxytoca), blaKPC-3 (one E. fergusonii), blaACT-36, and blaACC-2 (two K. oxytoca, four E. fergusonii, and two E. coli). The IncFIIs, IncF, IncFIB, IncK, IncP, and IncX replicons were detected among these beta-lactamase Enterobacteriaceae producers. The blaKPC-2, tetA, sul3, qnrB, and cmlA determinants were co-transferred by conjugation from K. oxytoca strain to E. coli J153, in association with IncK and IncF replicons. Our results support the implication of wild birds as a biological vector for carbapenemase, ESBL, and acquired-AmpC-producing Enterobacteriaceae.

Characterization of Beta-lactamases in Faecal Enterobacteriaceae Recovered from Healthy Humans in Spain: Focusing on AmpC Polymorphisms.

The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.

IncI1 plasmids carrying bla(CTX-M-1) or bla(CMY-2) genes in Escherichia coli from healthy humans and animals in Tunisia.

The objective was to determine the location of bla(CTX-M-1) and bla(CMY-2) genes in 33 Escherichia coli isolates previously obtained from healthy humans, pets, and food-producing animals in Tunisia, and to characterize the genetic lineages of isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE)-XbaI and multilocus sequence typing (MLST). Plasmids were analyzed by S1-PFGE, polymerase chain reaction-based replicon typing, and plasmid MLST. Conjugation experiments were performed. The bla(CTX-M-1) and bla(CMY-2) genes were studied by I-Ceu1-PFGE and S1-PFGE, and subsequent hybridization with specific probes. Eighteen different sequence types (STs) were identified among the 30 CTX-M-1-producing isolates, 5 of them being detected in 17 isolates (ST/phylogroup): ST57/D, ST155/B1, ST58/B1, ST10/A, and ST398/A. Most of the bla(CTX-M-1)-positive isolates had different PFGE profiles, with the exception of four human and pet isolates of lineage ST57 with related PFGE profiles (>80% identity). Three CMY-2-producing isolates were typed as ST58/B1, ST117/D, and ST3632/B2. The IncI1 replicon was detected in all the 33 E. coli studied isolates, in many cases in combination with other replicons: IncF, IncX, IncK, IncR, IncY, colE, or IncN. The bla(CTX-M-1) gene was transferred by conjugation in 22 of the 30 positive strains and was located into IncI1 plasmids (ST3-CC3); the bla(CMY-2) gene was located into a conjugative IncI1 plasmid (ST12) of 97 kb in one strain. One bla(CTX-M-1)-positive strain carried the qnrB19 gene in a 33 kb IncX plasmid. Diverse genetic lineages are detected in extended-spectrum beta-lactamase- and AmpC beta-lactamase-producing E. coli from different origins. The bla(CTX-M-1) and bla(CMY-2) genes were associated with conjugative IncI1 (ST3 and ST12, respectively) plasmids in E. coli strains from human and animal origin.

Emergence of a multiresistant KPC-3 and VIM-1 carbapenemase-producing Escherichia coli strain in Spain.

OBJECTIVES: To characterize the mechanisms involved in carbapenem resistance, as well as the genetic elements supporting their mobilization, in a multidrug-resistant Escherichia coli isolate. METHODS: The E. coli isolate was obtained from a patient with fatal urinary sepsis. Antimicrobial susceptibility testing was performed by the disc diffusion and agar dilution methods. The E. coli molecular type and phylogroup were determined using multilocus sequence typing and the triple PCR technique, respectively. PCR and sequencing were used for virulence and resistance genotype characterization. Plasmid content and gene location were analysed by S1-PFGE, I-Ceu1-PFGE and hybridization experiments. Transformation assays were performed. RESULTS: The E. coli strain, typed as ST448 and phylogroup B1, was resistant to all tested antibiotics except fosfomycin, tigecycline and tetracycline. The following resistance and virulence genetic structures were obtained: ISKpn7 + bla(KPC-3) + ISKpn6 linked to Tn4401; tnpR + aac(6')-Ib'-9 + aadA1 + bla(OXA-9) + tnpR + bla(TEM-1a) + tnpB + strB + strA + sul2; intI1 + bla(VIM-1) + aac(6')-Ib' + aphA15 + aadA1 + catB2 + qacEΔ1-sul1 + orf5; ISEcp1 + bla(CMY-2); IS26 + bla(SHV-12); aph(3')-I; aac(3)-IV; floR; catA; and fimA. Mutations in the ampC promoter (-18, -1 and +58) and substitutions in the GyrA (Ser-83→Leu and Asp-87→Asn) and ParC (Ser-80→Ile) proteins were observed. IncFII (ST2), IncA/C and ColE(TP) plasmids of 145.5, 87 and <2 kb, respectively, were found. The bla(VIM-1) gene was located in a non-typeable plasmid of >300 kb, and the bla(KPC-3) gene in the 145.5 kb IncFII plasmid. Transformant strains carried the IncFII and ColE(TP) plasmids, and the bla(KPC-3), bla(TEM-1a), bla(OXA-9), aadA1, aac(6')-Ib'-9, aac(3)-IV and floR genes. CONCLUSIONS: This is the first report of the co-production of KPC-3, VIM-1, SHV-12, OXA-9 and CMY-2 in a unique clinical multiresistant E. coli isolate. The dissemination of these genes on mobile genetic elements is alarming and complicates antimicrobial therapies.

First detection of CTX-M-1, CMY-2, and QnrB19 resistance mechanisms in fecal Escherichia coli isolates from healthy pets in Tunisia.

Our objective was to analyze the carriage rate of extended-spectrum ß-lactamase (ESBL)- and plasmidic AmpC ß-lactamase (pAmpC)-producing Escherichia coli isolates in fecal samples of healthy pets (dogs and cats) and to characterize the recovered isolates for the presence of other resistance genes and integrons. Eighty fecal samples of healthy pets were inoculated in MacConkey agar plates supplemented with cefotaxime (2 µg/mL) for cefotaxime-resistant (CTX(R)) E. coli recovery. CTX(R) E. coli isolates were detected in 14 of the 80 fecal samples (17.5%) and the following ß-lactamase genes (number of isolates) were detected: bla(CTX-M-1) (8), bla(CTX-M-1)+bla(TEM-1b) (3)(,) bla(CTX-M-1)+bla(TEM-1c) (1), bla(CTX-M-1)+bla(TEM-135) (1), and bla(CMY-2)+bla(TEM-1b) (1). The 14 E. coli were distributed into the phylogroups B1 (6 isolates), A (5), and D (3). The qnrB19 gene was detected in one CTX-M-1-producing strain of phylogroup D. Five isolates contained class 1 integrons with the following arrangements: dfrA17-aadA5 (2 isolates), dfrA1-aadA1 (1), and dfrA17-aadA5/ dfrA1-aadA1 (2 isolates). The virulence genes fimA and/or aer were detected in all CTX(R) strains. In this study, the pet population harbored ß-lactamase and quinolone resistance genes of special interest in human health that potentially could be transmitted to humans in close contact with them.

Changes in genetic lineages, resistance, and virulence in clinical methicillin-resistant Staphylococcus aureus in a Spanish hospital.

A total of 204 methicillin-resistant Staphylococcus aureus (MRSA) isolates were isolated in a Spanish hospital in two different periods (2001 and 2009). The percentages of MRSA isolates detected in 2001 and 2009 were 29 and 27 %, respectively. Genetic lineages, resistance mechanisms, and virulence traits were determined in these isolates. The most frequent detected lineage in both periods was S. aureus protein A (spa)-type t067, assigned to clonal complex (CC) 5 (CC5-t067), being more prevalent in 2001 (93 %) than in 2009 (71 %). The remaining CCs and spa-types detected were (%2001/%2009): CC5-t002 (0/5), CC8-t008 (1/16), CC8-t024 (0/1), CC8-t190 (0/3), CC8-t2849 (0/2), CC22-t032 (0/2), CC30-t012 (1/0), CC228-t109 (1/0), CC228-t1318 (2/0), and CC247-t051 (2/0). Most of the MRSA were isolated from wounds, representing 39 % in 2001 and 63 % in 2009. The emergence of MRSA CC8 isolates, mainly from wounds, seemed to occur in the second period. Resistance to (%2001/%2009) quinolones (99/87), aminoglycosides (98/88), macrolides (32/30), lincosamides (30/17), and tetracycline (2/1) was found in isolates in both periods. Trimethoprim-sulfamethoxazole resistance was detected only in 2001 (1 %), and chloramphenicol (1 %) and mupirocin resistance (11 %) were detected only in 2009. An association between staphylococcal enterotoxin gene profiles and CCs was detected in most of the cases. The egc-cluster was related to CC5, CC22, CC30, and CC228 and most of the CC8 isolates presented the sed, sej, and ser genes. Four tst-1-positive (CC5 and CC30) isolates were detected in 2001 and two lukS/F-PV-positive isolates were detected in 2009. Therefore, there is still a predominance of CC5-t067 in our region, although an increase of lineage CC8 was observed.

Epidemiological features, resistance genes, and clones among community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) isolates detected in northern Spain.

Twenty-nine community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) isolates were prospectively selected according to epidemiological criteria among 374 MRSA isolates collected in our laboratory during 2009-2010 in order to determine which community-associated MRSA (CA-MRSA) and healthcare-associated MRSA (HA-MRSA) clones are circulating in the community in northern Spain. PVL genes were detected in 5 strains (17.2%) that belonged to SCCmec type IV or V and to the agr group I (ST8 and ST2050), agr group II (ST121), and agr group III (ST30 and ST852). These strains were isolated from patients with different clinical manifestations such as urinary tract infection, abscess, or pneumonia, and most of them belonged to emergency department patients with no history of visits to General Practitioners (GPs) in the year before the isolation. We considered that the prevalence of CA-MRSA in community-onset isolates was low (17.2%). A high proportion of the CO-MRSA strains (58.6%) were ST125-MRSA-IVc (CC5), responsible for most of the infections caused by HA-MRSA strains in Spain. This endemic clone is also circulating in the community of northern Spain as we could demonstrate in this study. Antimicrobial resistance was found in spa type t067 isolates linked to the presence of ant(4')-Ia and msr(A). Most of the CO-MRSA isolates in this study corresponded to spa types more associated to the hospital environment, suggesting the interchange of genetic lineages of MRSA among community and hospital niches.