Emerging Antimicrobial-Resistant High-Risk Klebsiella pneumoniae Clones ST307 and ST147.

There is an enormous global public health burden due to antimicrobial-resistant (AMR) Klebsiella pneumoniae high-risk clones. K. pneumoniae ST307 and ST147 are recent additions to the family of successful clones in the species. Both clones likely emerged in Europe during the early to mid-1990s and, in a relatively short time, became prominent global pathogens, spreading to all continents (with the exception of Antarctica). ST307 and ST147 consist of multiple clades/clusters and are associated with various carbapenemases (i.e., KPCs, NDMs, OXA-48-like, and VIMs). ST307 is endemic in Italy, Colombia, the United States (Texas), and South Africa, while ST147 is endemic in India, Italy, Greece, and certain North African countries. Both clones have been introduced into regions of nonendemicity, leading to worldwide nosocomial outbreaks. Genomic studies showed ST307 and ST147 contain identical gyrA and parC mutations and likely obtained plasmids with blaCTX-M-15 during the early to mid-2000s, which aided in their global distribution. ST307 and ST147 then acquired plasmids with various carbapenemases during the late 2000s, establishing themselves as important AMR pathogens in certain regions. Both clones are likely underreported due to restricted detection methodologies. ST307 and ST147 have the ability to become major threats to public health due to their worldwide distribution, ability to cause serious infections, and association with AMR, including panresistance. The medical community at large, especially those concerned with antimicrobial resistance, should be aware of the looming threat posed by emerging AMR high-risk clones such as K. pneumoniae ST307 and ST147.

A Fatal Bacteremia Caused by Hypermucousviscous KPC-2 Producing Extensively Drug-Resistant K64-ST11 Klebsiella pneumoniae in Brazil.

We report a fatal bacteremia caused by Klebsiella pneumoniae in a 60-70-year-old patient from Brazil. The genomic analysis of three isolates (from blood culture, nasal and anal swabs) showed that the bacteremia was caused by a KPC-2 producing extensively drug-resistant K64-ST11 hypermucousviscous K. pneumoniae (hmKP) harboring several virulence and antimicrobial resistance genes. Although the isolates did not present virulence markers associated with hypervirulent K. pneumoniae (hvKP), they showed invasion and toxicity to epithelial Hep-2 cells; resistance to cell microbicidal mechanisms; and blood and human serum survival, evidencing their pathogenic potential. This study highlights the risk of infection caused by hmKp strains not characterized as hvKP as well as the clinical implications and difficulty of treatment, especially in elderly or immunocompromised patients.

Global Molecular Epidemiology of IMP-Producing Enterobacteriaceae.

International data on the molecular epidemiology of Enterobacteriaceae with IMP carbapenemases are lacking. We performed short-read (Illumina) whole-genome sequencing on a global collection of 38 IMP-producing clinical Enterobacteriaceae (2008 to 2014). IMP-producing Enterobacteriaceae (7 varieties within 11 class 1 integrons) were mainly present in the South Pacific and Asia. Specific blaIMP-containing integrons (In809 with blaIMP-4, In722 with blaIMP-6, and In687 with blaIMP-14) were circulating among different bacteria in countries such as Australia, Japan, and Thailand. In1312 with blaIMP-1 was present in Klebsiella pneumoniae from Japan and Citrobacter freundii from Brazil. Klebsiella pneumoniae (n = 22) was the most common species; clonal complex 14 (CC14) from Philippines and Japan was the most common clone and contained In1310 with blaIMP-26 and In1321 with blaIMP-6 The Enterobacter cloacae complex (n = 9) consisted of Enterobacter hormaechei and E. cloacae cluster III. CC78 (from Taiwan) containing In73 with blaIMP-8 was the most common clone among the E. cloacae complex. This study highlights the importance of surveillance programs using the latest molecular techniques for providing insight into the characteristics and global distribution of Enterobacteriaceae with blaIMP genes.

Dissemination of multidrug-resistant Acinetobacter baumannii genotypes carrying bla(OXA-23) collected from hospitals in Rio de Janeiro, Brazil.

The present study reports the dissemination of multidrug-resistant (MDR) OXA-23-producing Acinetobacter baumannii clones throughout hospitals in Rio de Janeiro, Brazil. A total of 110 imipenem-resistant A. baumannii isolates were obtained from January 2006 to September 2007 in eight hospitals. The modified Hodge test was performed to screen for carbapenemase production. Polymerase chain reaction (PCR) and DNA sequencing were performed for the detection of bla(IMP), bla(VIM), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58) and the class 1 integron. Isolates were typed by pulsed-field gel electrophoresis (PFGE) following digestion with ApaI. All the isolates were MDR and 96 (87.3%) produced the carbapenemase OXA-23. No isolates produced OXA-24, OXA-58 or the metallo-beta-lactamases IMP and VIM. The class 1 integron was absent in all isolates. The A. baumannii isolates were separated into five genotypes, with the highest prevalence of genotype A (71.8%) followed by genotype B (22.7%). Genotype A was present in seven hospitals, whilst genotype B had spread in five hospitals. The OXA-23-producing isolates belonged to all genotypes. The presence of MDR OXA-23-producing A. baumannii in different hospitals in Rio de Janeiro emphasises the need to control the use of carbapenems and to prevent the spread of these organisms in Rio de Janeiro.

Carbapenem-hydrolysing beta-lactamase KPC-2 in Klebsiella pneumoniae isolated in Rio de Janeiro, Brazil.

OBJECTIVES: The aim of this study was to characterize the KPC-type carbapenem-hydrolysing beta-lactamase, extended-spectrum beta-lactamases (ESBLs) and class 1 integrons among nosocomial Klebsiella pneumoniae isolated in Rio de Janeiro, Brazil. METHODS: MICs were determined and isolates were screened for ESBLs, metallo-beta-lactamases (MBLs) and class A carbapenemase-producing phenotypes. The main beta-lactamases resistance genes (bla(TEM), bla(SHV), bla(CTX-M), bla(KPC), bla(IMP) and bla(VIM)) and class 1 integrons were detected by PCR followed by DNA sequencing. The genetic relatedness of isolates was determined by PFGE. RESULTS: All K. pneumoniae isolates were positive for ESBL and class A carbapenemase production and negative for MBL production. All isolates were resistant to all beta-lactam antibiotics, ciprofloxacin and gentamicin, being susceptible only to tigecycline and polymyxin B. The bla(KPC-2), bla(CTX-M-1), bla(CTX-M-2), bla(CTX-M-8) and bla(SHV-11) genes were detected. PFGE analysis revealed two clonal types among KPC-producing isolates, both identified in the same hospital. CONCLUSIONS: Our findings should alert medical authorities to implement stringent methods for the detection and spread control of emerging KPC-2 carbapenemases in the hospital setting in Brazil.

dfrA25, a novel trimethoprim resistance gene from Salmonella Agona isolated from a human urine sample in Brazil.

OBJECTIVES: To describe a novel trimethoprim resistance gene, designated dfrA25, which was detected as a gene cassette within a class 1 integron in Salmonella Agona. METHODS: The gene was cloned into Escherichia coli MT102 and resistance to 10 different antimicrobial drugs was measured. A phylogenetic tree was constructed based on representative trimethoprim-resistance-mediating DfrA proteins retrieved from GenBank. Filter-mating experiments and Southern blots of plasmid preparations were performed with the donor and selected transconjugants. RESULTS AND CONCLUSIONS: dfrA25 encodes a dihydrofolate reductase of 157 amino acids with closest identity (85%) to dfrA5 dihydrofolate reductase. dfrA25 was located on a transferable plasmid (approximately 150 kb) that also harboured the tetracycline resistance gene tet(A).

Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil.

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED)--IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7%), S. sonnei (44.2%), S. boydii (2.3%), and S. dysenteriae (0.6%). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39%) and Northeast (34%) regions and the lowest rate in the South (3%) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90%), tetracycline (88%), ampicillin (56%), and chloramphenicol (35%). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.

Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil.

OBJECTIVES: To determine the occurrence of antimicrobial resistance genes and role of integrons among 135 antimicrobial-resistant Salmonella enterica from Brazil. METHODS: The presence of antimicrobial resistance genes, class 1 and 2 integrons and gene cassettes was analysed by PCR and sequencing. The genetic location of class 1 integrons was determined in 25 isolates by hybridization and plasmid transfer experiments. RESULTS: Fifty-five of the isolates were positive for class 1 integrons. Integron-positive isolates represented 17 different serovars and were mainly from human (n=28) and animal (n=13) sources. The gene cassette arrangements could be determined in 51 of the positive isolates, which harboured one [dfrA22, aadA1 or orf3 (putative trimethoprim resistance)], two [aadA1-dfrA1, aac(6')-Ib-orf1 (unknown function) or aacA4-aadA1], three [dfrA15b-cmlA4-aadA2, orf2 (unknown function)-dfrA5-orfD] or four [orf4-aacA4-blaOXA-30 (interrupted by an IS1 element)-aadA1] cassettes in their variable region. Only one isolate harboured a class 2 integron with the gene cassette array dfrA1-sat-aadA1. Several integron unrelated resistance genes were also detected in the isolates. Sulphonamide resistance was primarily mediated by sul2 and sul3, tetracycline resistance by tet(B) and tet(A), chloramphenicol resistance by catA1, streptomycin resistance by strA and ampicillin resistance by blaTEM. blaCTX and blaCMY-2 were found in cephalosporin-resistant isolates. Mating and hybridization experiments demonstrated that a high-molecular-weight plasmid mediated the gene transfer of integrons and additional resistance determinants. CONCLUSIONS: The present study revealed that integron-mediated resistance genes contributed to the multiresistance phenotype observed in the isolates, but most resistance genes were located outside the integron structure, as independent genes. However, they might be located on the same conjugative plasmid.

Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED) - IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7 percent), S. sonnei (44.2 percent), S. boydii (2.3 percent), and S. dysenteriae (0.6 percent). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39 percent) and Northeast (34 percent) regions and the lowest rate in the South (3 percent) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90 percent), tetracycline (88 percent), ampicillin (56 percent), and chloramphenicol (35 percent). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.

Caracterização molecular da resistência antimicrobiana em Salmonella spp e Shizella spp multiresistentes isoladas no Brasil / Molecular characterization of the antimicrobiana resistance in Salmonella spp and isolated multiresistant Shizella spp in Brazil

A presença de genes de resistência antimicrobiana e de integrons de classes 1 e 2 foi analisada em amostras de Shigella spp. (n=62) e Salmonella spp. (n=135) multirresistentes isoladas entre 1999 e 2003, no Brasil. Os principais perfis incluíram um espectro de resistência ao sulfametoxazol, trimetoprim, estreptomicina, espectinomicina, tetraciclina, cloranfenicol e ácido nalidíxico entre as Salmonella, e ao sulfametoxazol, trimetoprim, estreptomicina, espectinomicina, tetraciclina, ampicilina e cloranfenicol entre as Shigella. Em Shigella, o integron de classe 1 foi detectado em duas cepas, enquanto o de classe 2 em 56 (90,3 por cento) cepas. Todos os isolados positivos para integron de classe 2 possuíam um fragmento similar de 2214 bp contendo o arranjo de cassetes gênicos dfrA1-sat-aadA1. Genes codificantes de resistência ao cloranfenicol (catA1), tetraciclina (tet(A) e tet(B)) e ampicilina (blaOXA e blaTEM), foram observados nas cepas resistentes. Em Salmonella, 55 (40,7 por cento) cepas foram positivas para integrons de classe 1. As cepas integron-positivos ocorreram em 17 sorovares, isolados, principalmente, de fontes humana (n = 28) e animal (n = 13). Os arranjos dos cassetes gênicos foram determinados em 51 (93 por cento) das cepas, as quais continham um (dfrA22, aadA1 ou dfrA25), dois (aadA1-dfrA1,aac/(6)-Ib-orf1(função desconhecida) ou aacA4-aadA1), três (dfrA15b-cmlA4-aadA2, orf2 (função desconhecida)-dfrA5-orfD) ou quatro (orf4-aacA4-blaOXA-30 (interrompida por IS1)-aadA1) cassetes em sua região variável. Somente duas cepas continham integrons de classe 2 com o arranjo de cassetes gênicos dfrA1-sat-aadA1. Vários genes de resistência não relacionados aos integrons também foram detectados. A resistência à sulfonamida foi mediada por sul2 e sul3, à tetraciclina por tet(B) e tet(A), ao cloranfenicol por catA1, à estreptomicina por strA e à ampicilina por blaTEM, blaCTX e blaCMY-2 detectados entre as cepas resistentes às cefalosporinas. Experimentos de conjugação e hibridização demonstraram que um plasmídeo de alto peso molecular era o mediador da transferência de integrons e de genes de resistência adicionais. Investigações sobre a prevalência, distribuição e tipos de genes de resistência localizados em integrons são importantes para o monitoramento da resistência antimicrobiana e para a compreensão do desenvolvimento da resistência ao nível molecular.

Occurrence of integrons and resistance genes among sulphonamide-resistant Shigella spp. from Brazil.

OBJECTIVES: To determine the occurrence of class 1 and 2 integrons and antimicrobial resistance genes among sulphonamide-resistant Shigella strains isolated in Brazil during 1999-2003. METHODS: Sixty-two Shigella (Shigella flexneri, n = 47 and Shigella sonnei, n = 15) were tested against 21 antimicrobial agents. The presence of integrons classes 1 and 2 and antimicrobial resistance genes was investigated by PCR using specific primers. RESULTS: A total of eight antimicrobial resistance profiles were identified, with the profile of resistance to sulfamethoxazole, trimethoprim, spectinomycin, streptomycin and tetracycline being the most common among S. sonnei, and additionally to ampicillin and chloramphenicol among S. flexneri. Class 1 integrons were found in only two strains, whereas class 2 integrons were found in 56 (90.3%) of the strains. All class 2-positive strains had a similar fragment of 2214 bp harbouring a gene cassette array conferring resistance to trimethoprim, streptothricin and spectinomycin/streptomycin. The genes coding for resistance to chloramphenicol (catA1), tetracycline [tet(A) and tet(B)] and ampicillin (bla(OXA) and bla(TEM)), were detected in resistant strains. CONCLUSIONS: The detection of class 1 and 2 integrons and additional antimicrobial resistance genes allowed us to identify the most frequent antimicrobial resistance patterns of Shigella spp. isolated in Brazil.

[Resistance to antibiotics and heavy metals from Escherichia coli isolated from sea water and pluvial galeries]. / Resistência a antibióticos e a metais pesados de Escherichia coli isoladas de agua do mar e galerias pluviais.

OBJECTIVE: Test the resistance to antibiotics and heavy metals of E. coli strains isolated from storm sewer water and adjacent seawater samples from three beaches (Meio, Area Preta and Ponta Negra) in the city of Natal/RN/Brazil, and determine the association among those characteristics. METHODS: A total of 98 strains of E. coli, 50 from storm sewers and 48 from the seawater were analyzed resistance to several antimicrobials by disk diffusion and agar dilution and to heavy metals by dilution in plates with aqueous solutions of CuSO4 incorporated to Mueller Hinton agar in concentrations of 100, 150, 200 and 250 ig/mL and HgCl2 in concentrations of 5, 10, 20, 30, and 50 ig/mL. Standard strains were used as control. RESULTS: Among the twelve antimicrobials tested, 28 (28.5%) of E. coli strains showed resistance to different antimicrobials drugs to seven. The greatest resistance rate was to tetracycline (46.4%), ampicillin (39.3%) and cephalothin (32.1%), with the remainder (nitrophurantoine, nalidixic acid, sulfatomexazol-trimethoprin and chloramphenicol) at lower percentages. Among the heavy metals, all the strains (100%) were resistant to zinc and to copper in the largest concentration (250 ig/mL), and 18.4% were resistant to HgCl the 50 ig/mL. Ten (55.5%) of the E. coli strains resistant to Hg were associated to resistance to antibiotics. CONCLUSION: These results suggest the existence of extra chromosomal genes in E. coli strains isolated from storm sewer water and adjacent seawater, which encoders of the resistance to antibiotics and heavy metals.