Rats as sources of multidrug-resistant Enterobacteriaceae in animal production environments.

Rattus norvegicus and Rattus rattus are commensal pest rodents, considered reservoirs and vectors of zoonotic pathogens. In livestock farms, the wide use of antimicrobials and their release into the environment lead to high long-term residual concentrations, which may in turn lead to the occurrence of antimicrobial resistance (AMR). Farm environments serve as AMR sources, resulting in the transmission of antimicrobial-resistant bacteria and their AMR genes of livestock origin into wildlife. This study aimed to analyse the profile of enterobacteria carrying AMR determinants in rats captured in livestock farms to determine their potential vectors as for the spread of AMR. To this end, 56 rats (52 R. norvegicus and 4 R. rattus) were live-trapped on 11 farms (pig, dairy, poultry and mixed farms) located in central Argentina, from spring 2016 to autumn 2017. From 50 of the R. norvegicus individuals and three of the R. rattus individuals found in 10 of the farms, we isolated 53 Escherichia coli and five Salmonella strains. Susceptibility to antimicrobials, genotypic profiles, minimal inhibitory concentration of colistin and the presence of mcr-1 and genes encoding extended-spectrum ß-lactamase (ESBL) were determined. Of the 58 isolates not susceptible to different antimicrobial classes, 28 of the E. coli strains and two of the Salmonella strains were defined as multi-drug resistant (MDR). S. Westhampton and S. Newport recovered were not susceptible to ampicillin or all the cephems tested. One of the E. coli obtained showed resistance to colistin and harboured the mcr-1 gene, demonstrated by PCR and conjugation. In two ESBL-producing Salmonella isolated from rats, CTX-M-2 genes were responsible for the observed resistance to third-generation cephalosporins. The MDR E. coli isolates showed several different resistance patterns (23), although some of them were the same in different individuals and different farms, with six resistance patterns, evidencing the dispersion of strains. These findings suggest that rats play a role in the dissemination of AMR determinants between animal, humans and environmental reservoirs.

Prevalence of Antimicrobial Resistance and Clonal Relationship in ESBL/AmpC-Producing Proteus mirabilis Isolated from Meat Products and Community-Acquired Urinary Tract Infection (UTI-CA) in Southern Brazil.

The present study aimed to evaluate the prevalence of antimicrobial resistance and clonal relationships in Proteus mirabilis isolated from chicken meat, beef, pork, and community-acquired urinary tract infections (UTI-CA). Chicken meat isolates showed the highest multidrug resistance (MDR), followed by those from pork and UTI-CA, whereas beef had relatively few MDR strains. All sources had strains that carried blaCTX-M-65, whereas blaCTX-M-2 and blaCMY-2 were only detected in chicken meat and UTI-CA isolates. This indicates that chicken meat should be considered an important risk factor for the spread of P. mirabilis carrying ESBL and AmpC. Furthermore, ESBL/AmpC producing strains were resistant to a greater number of antimicrobials and possessed more resistance genes than non-producing strains. In addition, the antimicrobial resistance genes qnrD, aac(6')-Ib-cr, sul1, sul2, fosA3, cmlA, and floR were also found. Molecular typing showed a genetic similarity between chicken meat and UTI-CA isolates, including some strains with 100% similarity, indicating that chicken can be a source of P. mirabilis causing UTI-CA. It was concluded that meat, especially chicken meat, can be an important source of dissemination of multidrug-resistant P. mirabilis in the community.

Genetic Characterization of CTX-M-2-Producing Klebsiella pneumoniae and Klebsiella oxytoca Associated With Bovine Mastitis in Japan.

CTX-M-2-producing Klebsiella oxytoca (K. oxytoca) has not received much attention in animal husbandry compared with Klebsiella pneumoniae (K. pneumoniae), a major reservoir of extended-spectrum ß-lactamase (ESBL) genes. Bacteriological examinations of 1,466 mastitic milk samples between October 2012 and December 2014 were conducted. Ninety-five K. pneumoniae isolates (total prevalence: 6.5%) and 81 K. oxytoca isolates (total prevalence: 5.5%) were obtained. Seventeen K. pneumoniae isolates obtained from 15 animals reared on 11 farms and 9 K. oxytoca isolates obtained from 9 animals reared on the same farm were phenotypically confirmed to be ESBL producers. All nine ESBL-producing K. oxytoca isolates were obtained from one farm between June and November 2013 and related to a significantly (p < 0.05) higher monthly prevalence of mild mastitis (in June, August, September, October, and November 2013). Pulsed-field gel electrophoresis (PFGE) patterns of ESBL-producing K. pneumoniae isolates were distinguished from each other by more than 6-band differences except for two isolates from two animals, whereas all nine K. oxytoca isolates showed an identical PFGE pattern. Transferability of the bla CTX-M-2 gene was found in 14 K. pneumoniae and 9 K. oxytoca isolates by conjugation analysis. Of these isolates, the bla CTX-M-2 gene was detected on plasmids belonging to the incompatibility (Inc) groups P and N derived from five K. pneumoniae and nine K. oxytoca isolates, respectively, although the plasmids from the remaining nine K. pneumoniae were untypeable. All the transconjugants exhibited elevated minimum inhibitory concentrations of ampicillin, cefotaxime, and ceftiofur compared with those in the wild-type, recipient strain. Restriction fragment length polymorphism analysis demonstrated that the IncN plasmids extracted from eight of nine transconjugants, which received resistance against ß-lactams from K. oxytoca, showed an identical DraI digestion pattern. These results suggest that the CTX-M-2-producing K. oxytoca strain with the above-mentioned characteristics may have clonally spread within a farm, whereas the bla CTX-M-2 gene in K. pneumoniae possibly disseminated among the farms through different plasmids. Thus, monitoring of ESBL genes, including the bla CTX-M-2 gene, among causative agents of bacterial mastitis in cows can help to develop relevant treatments and control practices.

Description of novel resistance islands harbouring blaCTX-M-2 in IncC type 2 plasmids.

OBJECTIVES: We sequenced two IncA/C plasmids harbouring blaCTX-M-2 in Klebsiella pneumoniae clinical isolates and compared their antibiotic resistance islands. METHODS: Transconjugants were obtained from two clinical K. pneumoniae isolates harbouring blaCTX-M-2. Plasmid DNA from transconjugants underwent short-read whole-genome sequencing, reads were assembled, and gaps were closed by PCR and sequencing. Determination of plasmid replicons, antibiotic resistance genes, identification and characterisation of insertion sequence (IS) elements, and comparison with publicly available plasmid sequences were performed. RESULTS: blaCTX-M-2 was located in a complex class 1 integron In35::ISCR1::blaCTX-M-2, inserted in two different transposons designated Tn7057 and Tn7058, that reside in the resistance islands of plasmids pUR-KP0923 and pUR-KP1025, respectively. The general modules of both transposons were In35::ISCR1::blaCTX-M-2-Tn1000-like-Tn2*-ISKpn11-12-13 variable module-ΔTn21. In Tn7057 there was ΔIS10R-catA2 associated with an additional ISKpn13. Both plasmids belonged to IncC type 2 and ST3. pUR-KP0923 was 167 138 bp in length and had a 37 926-bp resistance island at position 4 (RI-4). Plasmid pUR-KP1025 was 168 128 bp with a RI-4 of 36 222 bp. CONCLUSION: This report describes the molecular nature of two transposons (Tn7057 and Tn7058) harbouring blaCTX-M-2 that reside in IncC type 2 ST3 plasmids. These transposons mediate resistance to oxyimino-cephalosporins, gentamicin and, in the case of Tn7057, chloramphenicol. CTX-M-2 is an important extended-spectrum ß-lactamase (ESBL) to South American epidemiology. It is remarkable that despite being only two plasmid sequences, the information revealed here could contribute to a better understanding of the resistance islands from IncC type 2 plasmids.

Characterization of antibiotic resistance profiles in Pseudomonas aeruginosa isolates from burn patients.

OBJECTIVE: To investigate the prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) producing extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria. METHODS: Between April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR). RESULTS: Among the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for six groups of bla genes encoding ESBL-type enzymes, namely blaCTX-M2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Out of the 28 ESBL-producing strains, 23 (82.14%) were blaCTX-M2 positive; 18 (38.29%) were blaPER positive, and 16 (34.04%) were blaTEM positive, while 5 (17.9%) were co-harboring blaCTX-M2, blaTEM, and blaPER genes. The blaSHV, blaVEB, and blaGES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the blaNDM-1, blaIMP, blaVIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the oprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 11 distinct PA clones among the blaCTX-M2 positive strains. CONCLUSION: This is the first study to report the presence of CTX-M2-producing PA in the North Africa region and the first to detect blaCTX-M2-positive and blaPER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains to this part of the world. Identification of bacterial genotypic alterations that confer antibiotic resistance is critical in determining the most effective antimicrobial strategies to be employed. Therefore, our findings could potentially facilitate clinical decision making regarding the antibiotics of choice for the treatment of burn patients that suffer from PA infections in Algeria.

Simultaneous Carriage of mcr-1 and Other Antimicrobial Resistance Determinants in Escherichia coli From Poultry.

The use of antimicrobial growth promoters (AGPs) in sub-therapeutic doses for long periods promotes the selection of resistant microorganisms and the subsequent risk of spreading this resistance to the human population and the environment. Global concern about antimicrobial resistance development and transference of resistance genes from animal to human has been rising. The goal of our research was to evaluate the susceptibility pattern to different classes of antimicrobials of colistin-resistant Escherichia coli from poultry production systems that use AGPs, and characterize the resistance determinants associated to transferable platforms. E. coli strains (n = 41) were obtained from fecal samples collected from typical Argentine commercial broiler farms and susceptibility for 23 antimicrobials, relevant for human or veterinary medicine, was determined. Isolates were tested by PCR for the presence of mcr-1, extended spectrum ß-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) coding genes. Conjugation and susceptibility patterns of the transconjugant studies were performed. ERIC-PCR and REP-PCR analysis showed a high diversity of the isolates. Resistance to several antimicrobials was determined and all colistin-resistant isolates harbored the mcr-1 gene. CTX-M-2 cefotaximase was the main mechanism responsible for third generation cephalosporins resistance, and PMQR determinants were also identified. In addition, co-transference of the qnrB determinant on the mcr-1-positive transconjugants was corroborated, which suggests that these resistance genes are likely to be located in the same plasmid. In this work a wide range of antimicrobial resistance mechanisms were identified in E. coli strains isolated from the environment of healthy chickens highlighting the risk of antimicrobial abuse/misuse in animals under intensive production systems and its consequences for public health.

Draft genome of a ST443 mcr-1- and blaCTX-M-2-carrying Escherichia coli from cattle in Brazil.

OBJECTIVES: Colistin is used in Brazil for the treatment of food-producing animals. The colistin resistance gene mcr-1 has already been reported from chicken and swine in this country. Here we report the draft genome of an Escherichia coli isolate presenting both an extended-spectrum ß-lactamase (ESBL) gene and the mcr-1 gene in a healthy cow in Brazil. METHODS: Whole genomic DNA from E. coli E12 was extracted and 2× 150-bp paired-end reads were generated using Illumina sequencing technology. De novo genome assembly was performed using SPAdes v.3.11 and the draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Further analyses were performed using Center for Genomic Epidemiology databases. Southern blots were performed to characterise plasmid location. RESULTS: The 5024393-bp genome displayed several resistance genes, including the mcr-1 and blaCTX-M-2 genes. These two genes were located on different plasmids (mcr-1 on an IncX4 plasmid and blaCTX-M-2 on an IncF plasmid). CONCLUSION: The genome sequence reported here can be compared with previously published genomes for mcr-1-producing isolates. This will ultimately help to understand the routes of dissemination of the resistance genes.

A multi-centre prospective evaluation of the Check-Direct ESBL Screen for BD MAX as a rapid molecular screening method for extended-spectrum beta-lactamase-producing Enterobacteriaceae rectal carriage.

OBJECTIVE: A multiplex extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) quantitative polymerase chain reaction (qPCR), performed directly on rectal swabs, was compared with a culture-based protocol to study the discrepancies between the two methods, and identify existing challenges to apply this assay in routine clinical practice. The secondary objective was to assess the performance of the qPCR. MATERIALS AND METHODS: In two Dutch teaching hospitals, 573 rectal swabs were collected prospectively. Culture with additional testing with the Check-MDR CT103XL (Check-Points) was compared with the Check-Direct ESBL Screen for BD MAX (Check-Points), which detects the presence of the ESBL gene families CTX-M1, CTX-M2, CTX-M9 and SHV2/5-ESBL. The culture-based protocol (with Brilliance agar) was considered as the gold standard to assess the performance of the qPCR. RESULTS: Of the 573 rectal swabs, 74 (12.9%) were culture-positive. Eighty-four (14.7%) were qPCR-positive. There were eight culture-positive/qPCR-negative discrepancies and 18 culture-negative/qPCR-positive discrepancies. Sensitivity and specificity of qPCR vs culture were 87.7% [95% confidence interval (CI) 79.7-95.7] and 96.3% (95% CI 94.6-98.0), respectively. CONCLUSION: The Check-Direct ESBL Screen for the BD MAX is an easy-to-perform, quick molecular diagnostic test with the potential to significantly speed up screening for rectal ESBL-E carriage. Discrepancies were observed between the culture-based protocol and the qPCR in 4.5% of tested samples. Existing challenges for implementing qPCR are its limited sensitivity, the need for thorough knowledge of the local ESBL-E genes, and interpretation of culture-negative but qPCR-positive samples. It is believed that the limited sensitivity of qPCR could be optimized by including blaTEM as a molecular target, and improving the limit of detection.

Regional Spread of CTX-M-2-Producing Proteus mirabilis with the Identical Genetic Structure in Japan.

AIMS: In this study, we analyzed the molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Proteus mirabilis isolates collected from the central region of Japan. MATERIALS AND METHODS: Between 2005 and 2012, 820 clinical P. mirabilis isolates were obtained from ten acute care hospitals in Japan. We characterized ESBL confirmatory test-positive isolates by sequencing the ESBL genes and their flanking regions, detecting plasmid replicons, and performing pulsed-field gel electrophoresis (PFGE). RESULTS: Ninety-six isolates (12%) were positive according to the ESBL confirmatory test; all these isolates possessed blaCTX-M-2 with the same flanking structure of upstream ΔISEcp1 and a downstream region identical to downstream blaKLUA-1. IncT was the prevalent, and only, replicon found in 63 isolates. PFGE analysis detected eight clusters with more than one isolate, among which three included 56 isolates and six included isolates from multiple hospitals. CONCLUSION: CTX-M-2-producing P. mirabilis with an identical genetic structure flanking blaCTX-M-2 is dominant in this Japanese region, and there is evidence for the clonal spread of isolates.

"Distribution and functional identification of complex class 1 integrons".

The emergence of extended-spectrum ß-lactamases and plasmid-mediated resistance to quinolones has been previously found to be associated with the dissemination of complex class 1 integrons in Argentina. In this study, we analyzed their distribution through time and evaluated the functionality of the Orf513 protein, which is the putative recombinase of the ISCR1 mobile element. We investigated the presence of the orf513, blaCTX-M-2, dfrA3b, qnrB10 and blaDHA-1 genes by PCR and DNA sequencing as well as their linkage to class 1 integrons in 451 non-epidemiologically related nosocomial strains resistant to at least one expanded-spectrum cephalosporin and to one aminoglycoside, isolated between 1989 and 2010 from 7 hospitals from Buenos Aires City. The epidemiology of complex class 1 integrons was found to be notably different among fermenting (94/171) and non-fermenting clinical bacilli isolates (1/280). The ISCR1::qnrB10 positive isolates were found since 1993, confirming its presence in clinical isolates more than a decade before its first description. As expected, In35::ISCR1::blaCTX-M-2 was the most common complex class 1 integron among Enterobacteriaceae isolates, particularly in Proteus mirabilis. Experimental analysis corroborated the activity of the Orf513 protein, which was found to bind specific DNA sequences containing the previously suggested oriIS region. These findings showed the high dispersion and maintenance of complex class 1 integrons across time in our nosocomial isolates. The contribution of the ISCR1 mobile element to multidrug resistant phenotypes is significant due to its sustained association to class 1 integrons.

Caracterização de beta-lactamases de espectro ampliado (ESBLs), genes de resistência aos antimicrobianos e conteúdo plasmidial em cepas de Escherichia coli e Salmonella spp. não tifóides isoladas do ambiente hospitalar e da comunidade / Characterization of extended spectrum Beta-lactamase (ESBLs), antimicrobial resistance genes, and plasmid content in Escherichia coli and Salmonella spp. isolates recovered from hospital and community

Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2)...

ESBL-producing Enterobacteriaceae have been described in hospitals and in the community worldwide. In Brazil, ESBL-producing Enterobacteriaceae have also emerged as an important cause of infections, being CTX-M enzymes the most prevalent ESBLs. The objective of this study was to analyze different genotypic aspects related to expression of antimicrobial resistance in isolates of Escherichia coli and Salmonella spp., such as: diversity of ESBLs, antibiotic resistance genes and plasmid content. Epidemiological features of ESBL-producing isolates were also investigated. We studied 88 isolates of enterobacteria, 43 E. coli and 45 Salmonella serotypes of hospital and community (mainly food) origin, isolated in the city of Rio de Janeiro. ESBL expression was observed in seven E. coli isolates (7/43; 16,3%) and in one Salmonella Typhimurium (1/45; 2,3%) and the enzymes identified as CTX-M variants and SHV-5, respectively. Among the E. coli isolates, CTX-M-2 was the most frequent (n=4), being detected in isolates recovered from rectal swabs of hospitalized patients, whereas CTX-M-59 (a CTX-M-2-variant) (n=1) and CTX-M-9 (n=2) were identified in E. coli isolated from clinical specimens. SHV-5-producing S. Typhimurium was isolated from the hospital environment (infant formula). CTX-M-producing E. coli belonged to different phylogenetic groups (A, B1, D) and STs (ST34, ST69, ST101), being blaCTX-M genes were identified in IncA/C plasmids of approximately 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) or 80 kb (blaCTX-M-2)...

Detección del gen codificante de la metalo-ß-lactamasa VIM-2 en un integrón de clase 1 asociado con el gen blaCTX-M-2 en un aislamiento clínico de Pseudomonas aeruginosa en el Uruguay: primera comunicación

Con el fin de analizar la presencia de metalo-ß-lactamasas en nuestro medio, se incluyeron en este estudio aislamientos de Pseudomonas aeruginosa causantes de infecciones nosocomiales en un centro hospitalario del Uruguay, en el período comprendido entre abril y setiembre de 2008. En un aislamiento se detectó la presencia del gen codificante de la metalo-ß-lactamasa VIM-2 asociado a un integrón de clase 1 y del gen codificante de una ß-lactamasa de espectro extendido CTX-M-2. Esta es la primera comunicación de la presencia de los genes blaCTX-M-2 y blaVIM-2 en un mismo aislamiento de P. aeruginosa. A pesar de que las carbapenemasas ya han sido ampliamente documentadas en varias partes del mundo, esta es la primera comunicación de una metalo-ß-lactamasa adquirida con actividad carbapenemasa en bacterias patógenas encontradas en el Uruguay.

VIM-2 metallo-ß-lactamase gen detection in a class 1 integron associated to blaCTX-M-2 in a Pseudomonas aeruginosa clinical isolate in Uruguay: first communication. In order to analyze the presence of metallo-ß-lactamase in our country, we included in this study Pseudomonas aeruginosa isolates causing nosocomial infections in a hospital from Uruguay. The presence of a metallo-ß-lactamase VIM-2 in a class 1 integron and of an extended spectrum -lactamase CTX-M-2 was detected in one isolate. This is the first report of both genes, blaCTX-M-2 and blaVIM-2,in the same P. aeruginosa isolate. Although carbapenemases have been extensively documented in the world, this is the first report of an acquired metallo-ß-lactamase with carbapenemase activity in pathogenic bacteria in Uruguay.