Beta-Lactam Antibiotic Resistance Genes in the Microbiome of the Public Transport System of Quito, Ecuador.

Multidrug-resistant bacteria present resistance mechanisms against ß-lactam antibiotics, such as Extended-Spectrum Beta-lactamases (ESBL) and Metallo-ß-lactamases enzymes (MBLs) which are operon encoded in Gram-negative species. Likewise, Gram-positive bacteria have evolved other mechanisms through mec genes, which encode modified penicillin-binding proteins (PBP2). This study aimed to determine the presence and spread of ß-lactam antibiotic resistance genes and the microbiome circulating in Quito's Public Transport (QTP). A total of 29 station turnstiles were swabbed to extract the surface environmental DNA. PCRs were performed to detect the presence of 13 antibiotic resistance genes and to identify and to amplify 16S rDNA for barcoding, followed by clone analysis, Sanger sequencing, and BLAST search. ESBL genes blaTEM-1 and blaCTX-M-1 and MBL genes blaOXA-181 and mecA were detected along QPT stations, blaTEM being the most widely spread. Two subvariants were found for blaTEM-1, blaCTX-M-1, and blaOXA-181. Almost half of the circulating bacteria found at QPT stations were common human microbiota species, including those classified by the WHO as pathogens of critical and high-priority surveillance. ß-lactam antibiotic resistance genes are prevalent throughout QPT. This is the first report of blaOXA-181 in environmental samples in Ecuador. Moreover, we detected a new putative variant of this gene. Some commensal coagulase-negative bacteria may have a role as mecA resistance reservoirs.

Longitudinal Study of Dynamic Epidemiology of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Pigs and Humans Living and/or Working on Pig Farms.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales have been increasingly isolated from pigs, highlighting their potential for transmission to humans living and/or working within pig farms. As longitudinal data on the prevalence and the molecular characteristics of such isolates from the high-risk farming population remain scarce, we performed a long-term study on 39 Dutch pig farms. Fecal samples from pigs, farmers, family members, and employees were collected during four sampling occasions with a 6-month period. The presence of ESBL-producing Enterobacterales and their molecular characteristics (ESBL gene, plasmid, and sequence types) were determined by standard methods. Data on personal and farm characteristics were collected using questionnaires. ESBL-producing Escherichia coli was present in pigs at least once for 18 of 39 farms and in 17 of 146 farmers, family members, and/or employees. Among these 417 E. coli isolates, blaCTX-M-1 was the most frequently observed ESBL gene in pigs (n = 261) and humans (n = 25). Despite the great variety in plasmid (sub)types and E. coli sequence types (STs), we observed genetic similarity between human- and pig-derived isolates in (i) ESBL gene, plasmid (sub)type, and ST, suggesting potential clonal transmission in seven farms, and (ii) only ESBL gene and plasmid (sub)type, highlighting the possibility of horizontal transfer in four farms. Five pig farmers carried ESBL producers repeatedly, of whom two carried an identical combination of gene, plasmid (sub)type, and ST over time. Human ESBL carriage was associated with both presence of ESBL producers in pigs and average number of hours working on the pig farm per week, while prolonged human carriage was observed only incidentally. IMPORTANCE Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a public health hazard due to reduced therapeutic options for the treatment of infections. Although direct contact with pigs is considered a risk factor for human ESBL-producing E. coli carriage through occupational exposure, nationwide data regarding the occurrence of such isolates among pigs and humans living and/or working on farms remain scarce. Therefore, we determined (i) the longitudinal dynamics in prevalence and molecular characteristics of ESBL-producing E. coli in Dutch pig farmers and their pigs over time and (ii) the potential transmission events between these reservoirs based on genetic relatedness and epidemiological associations in longitudinal data. Our data suggesting the possibility of clonal and horizontal dissemination of ESBL-producing Escherichia coli between pigs and pig farmers can be used to inform targeted intervention strategies to decrease the within-farm human exposure to ESBL-producing E. coli.

Corrigendum: bla CTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].

Corrigendum: bla CTX-M-1 /IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].

Prevalence of ESßL, AmpC and Colistin-Resistant E. coli in Meat: A Comparison between Pork and Wild Boar.

A global increase in Escherichia coli (E. coli) resistant to cephalosporins (extended-spectrum ß-lactamases (ESßLs) and AmpC ß-lactamases) has been recorded in the last 20 years. Similarly, several studies have reported the spread of colistin resistance in Enterobacteriaceae isolated from food and the environment. The aim of the present study was to evaluate the prevalence of ESßL, AmpC and colistin-resistant E. coli isolated from pork and wild boar meat products in the Emilia Romagna region (North Italy). The isolates were analysed phenotypically (considering both resistant and intermediate profiles) and genotypically. The prevalence of genotypically confirmed ESßL and AmpC E. coli was higher in pork meat products (ESßL = 11.1% vs. AmpC = 0.3%) compared to wild boar meat (ESßL = 6.5% vs. AmpC = 0%). Intermediate profiles for cefotaxime (CTX) and ceftazidime (CAZ) were genotypically confirmed as ESßL in pork meat isolates but not for wild boar. Four E. coli from wild boar meat were resistant to colistin but did not harbour the mcr-1 gene. E. coli isolated from wild boar meat seem to show aspecific antimicrobial resistance mechanisms for cephalosporins and colistin. The prevalence of resistant isolates found in wild boar is less alarming than in pork from farmed domestic pigs. However, the potential risk to consumers of these meat products will require further investigations.

blaCTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

Escherichia coli carrying blaCTX-M-1 mediating resistance to extended-spectrum cephalosporins was recently described as a new genotype in Norwegian broiler production. The aim of this study was to characterize these isolates (n = 31) in order to determine whether the emergence of the genotype was caused by clonal expansion or horizontal dissemination of blaCTX-M-1-carrying plasmids. All included isolates were subjected to whole genome sequencing. Plasmid transferability was determined by conjugation, and plasmid replicons in the transconjugants were described using PCR-based replicon typing. Plasmid sizes were determined using S1 nuclease digestion. Plasmids in a subset of strains were reconstructed and compared to plasmids from broiler production in other European countries. The isolates belonged to nine different sequence types (STs), with the largest group being ST57 (n = 12). The vast majority of blaCTX-M-1-carrying plasmids were conjugative. All transconjugants were positive for the IncI1-Iγ replicon, and several also harbored the IncFIB replicon. Highly similar plasmids were present in different E. coli STs. Additionally, high similarity to previously published plasmids was detected. A reconstructed plasmid from an ST57 isolate harbored both IncI1-Iγ and IncFIB replicons and was considered to be co-integrated. The presence of one large plasmid was confirmed by S1 nuclease digestion. Our results show that dissemination of blaCTX-M-1 in Norwegian broiler production is due to both clonal expansion and horizontal transfer of plasmids carrying blaCTX-M-1. The blaCTX-M-1/IncI1-Iγ plasmids grouped into two main lineages, namely clonal complex (CC)-3 and CC-7. The genetic diversity at both strain and plasmid level indicates multiple introductions to Norway. We also show that the blaCTX-M-1 plasmids circulating in Norwegian broiler production are highly similar to plasmids previously described in other countries.

Identification of Genes Essential for Antibiotic-Induced Up-Regulation of Plasmid-Transfer-Genes in Cephalosporin Resistant Escherichia coli.

Bacterial conjugation is one of the most important mechanisms for spread of antibiotic resistance among bacteria. We have previously demonstrated that cefotaxime (CTX) exposure up-regulates expression of Type-IV conjugation transfer genes, and that this leads to increased transfer of a bla CTX-M- 1 encoding IncI1 resistance plasmid pTF2 in Escherichia coli. To elucidate the underlying mechanisms, a search for genes that are essential for the up-regulated expression of the transfer (tra) genes in the presence of CTX was undertaken. We constructed a reporter gene-fusion strain MG1655/pTF2 ΔtraF:lacZ where the promoter region of the traF-gene of the plasmid pTF2 was fused with a lacZ on the native plasmid. Random mutagenesis mediated by Tn5 transposon was carried out in the strain, and seven genes (rfaH, yhiN, waaP, waaQ, gnd, pgl, and ISEcp1) were identified where insertion prevented CTX-induced up regulation of traF. Site-specific mutagenesis was carried out, and for all seven mutants, gene deletions abolished the CTX induced up-regulation of traF, and the increased conjugation transfer of the plasmid in the presence of CTX was no longer observed. In addition, the deletion of the genes also abolished CTX induced expression of the bla CTX-M- 1 gene. Our results suggested that through CTX induced induction of the identified genes, bla CTX-M- 1 expression increased, which led to up-regulation of traF and plasmid transfer. These data reveal that a number of chromosomally encoded genes contribute to the antibiotic induced up-regulation of the conjugation machinery of plasmids, and such genes may be future targets to prevent antibiotic induced spread of resistance plasmids.

Is blaCTX-M-1 Riding the Same Plasmid Among Horses in Sweden and France?

A predominance of the blaCTX-M-1/IncHI1 plasmid combination in horses has been reported in Czech-Republic, Denmark, and The Netherlands. To clarify a possible specific plasmid epidemiology of blaCTX-M-1 in horses in a European perspective, a collection of 74 extended-spectrum beta-lactamase-producing Escherichia coli recovered from diseased horses in France and Sweden during the period 2009-2014 was investigated in respect of their genetic relatedness, plasmid content, and molecular features. Overall, 80% of E. coli isolates from diseased horses harbored blaCTX-M-1 on large IncHI1 plasmids with plasmid sequence type (pST) 2 and pST9 more prevalent in Sweden and France, respectively. In French isolates, IncI1/pST3 plasmids were also identified. The CTX-M-1-producing E. coli belonged principally to the clonal complex 10. ST641, together with its single locus variant, and ST1730 constituted two other major groups. The rep-PCR clustering highlighted a clonal dissemination of E. coli CTX-M-1 producers in different regions of the same country and during several years. The STs found in our isolates were also reported in The Netherlands, suggesting a common source of contamination in Europe, although only further cooperative investigation will clarify this issue. On the other hand, the spread of IncI1/pST3 plasmids among horses constitutes another worrisome issue considering their successful spread in other animal hosts such as chicken or bovines. Monitoring evolution and propagation of antimicrobial resistance in equine environment is a priority to avoid further propagation of antimicrobial resistance mechanisms threatening human and animal health.

Molecular characterization of antimicrobial resistance in clinical Shigella isolates during 2014 and 2015: trends in South India.

BACKGROUND: Shigella species are an important cause of acute diarrheal disease worldwide. This study describes the prevalence of Shigella spp. serotypes and their resistance profile in Vellore, South India from 2014 to 2015. METHODS: From 2014 to 2015, 338 Shigella strains were isolated from stool samples at Christian Medical College, Vellore, India. Identification and serotyping was carried out using standard protocols. Antimicrobial susceptibility testing was done against commonly used antibiotics. Multidrug resistance was detected in 157 isolates. A subset of 73 isolates was randomly characterized further for acquired antimicrobial resistance genes in this study. RESULTS: The resistance profile of the study isolates varied by species and year. S. sonnei isolates were 100% resistant to all tested antibiotics in 2014, whereas in 2015, resistance was found for AMP-NAL-TAX-SXT-FIX. The resistance phenotypes among S. flexneri isolates for the year 2014 and 2015 were AMP-SXT-NAL-NOR-FIX-TAX and AMP-NAL-SXT-TAX-NOR-FIX respectively. Screening for antimicrobial resistance genes in S. flexneri found dhfr1A, sulII, blaOXA, blaTEM, blaCTX-M-1,qnrB, qnrS and AmpC genes while S. sonnei were found to have only dhfr1A, sulII, blaCTX-M-1 and qnrS genes respectively. Antimicrobial resistance genes were predominantly seen in AMP-SXT-NAL and AMP-SXT-NAL-NOR resistance phenotypes. CONCLUSION: Shigella prevalence of 4.8% to 4.6% was documented between the years 2014 to 2015 in this study. We show evidence that resistance to commonly used antibiotics continues to increase among Shigella spp. in South India. The presence of qnrS and blaCTX-M-15 in the study isolates further indicates the threat of spreading resistance to quinolones and third-generation cephalosporins.

Prevalence of the bla CTX-M-1 group and their transferability in resistant clinical isolates of Salmonella serogroups from several hospitals of Tehran.

BACKGROUND AND OBJECTIVES: Salmonella is an important food-borne pathogen in humans. Strains of Salmonella spp. that producing extended-spectrum ß-Lactamases have become a concern in medicine regarding both antimicrobial treatment and infection control program. The objective of this study was to describe the antibiotic susceptibility, ESBL production and determining the prevalence of the bla CTX-M-1 group among clinical isolates of Salmonella spp. MATERIALS AND METHODS: A total of 110 Salmonella isolates collected from four Tehran hospitals during May 2012 and April 2013. The specific monovalan Salmonella antisera were used for serogrouping of Salmonella isolates. Antibacterial susceptibility was determined by disk diffusion and ESBL phenotype was confirmed by combination disk method. The bla CTX-M-1 group was identified by PCR with specific primers. The transferability of the bla CTX-1 group was tested by conjugation with broth matting method. RESULTS: The prevalence of Salmonella serogroups consist of 56.4% serogroup D, 13.6 % serogroup C, 10 % serogroup B, and 1.8 % serogroup A and 18.2% other serogroups. Maximal resistance in Salmonella isolates was noticed against trimethoprim-sulfamethoxazole (63.6%) and nalidixic-acid (47/3%). All isolates were susceptible to imipenem and ciprofloxacin. Four isolates (3.6%) showed ESBLs phenotype. All Salmonella spp. that produce ESBls have bla CTX-1 genes group. A conjugative plasmid containing bla CTX-1 group was found in one Salmonella isolate. CONCLUSION: This study demonstrates the predominant presence of the gene encoding CTX-M-1 group among ESBLs producing of Salmonella spp. They can transmit to bacteria of this genus or even other genera of enteric bacteria.

Occurrence of bla CTX-M-1, qnrB1 and virulence genes in avian ESBL-producing Escherichia coli isolates from Tunisia.

Avian ESBL-producing Escherichia coli isolates have been increasingly reported worldwide. Animal to human dissemination, via food chain or direct contact, of these resistant bacteria has been reported. In Tunisia, little is known about avian ESBL- producing E. coli and further studies are needed. Seventeen ESBL-producing Escherichia coli isolates from poultry feces from two farms (Farm 1 and farm 2) in the North of Tunisia have been used in this study. Eleven of these isolates (from farm 1) have the same resistance profile to nalidixic acid, sulfonamides, streptomycin, tetracycline, and norfloxacine (intermediately resistant). Out of the six isolates recovered from farm 2, only one was co-resistant to tetracycline. All isolates, except one, harbored bla CTX-M-1 gene, and one strain co-harbored the bla TEM-1 gene. The genes tetA and tetB were carried, respectively, by 11 and 1 amongst the 12 tetracycline-resistant isolates. Sulfonamides resistance was encoded by sul1, sul2, and sul3 genes in 3, 17, and 5 isolates, respectively. The qnrB1 was detected in nine strains, one of which co-harbored qnrS1 gene. The search for the class 1 and 2 integrons by PCR showed that in farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, class 2 was not detected. Only one gene cassette arrangement was demonstrated in the variable regions (VR) of the 10 int2-positive isolates: dfrA1- sat2-aadA1. The size of the VR of the class 1 integron was approximately 250 bp in one int1-positive isolate, whereas in the second isolate, no amplification was observed. All isolates of farm 1 belong to the phylogroup A (sub-group A0). However, different types of phylogroups in farm 2 were detected. Each of the phylogroups A1, B22, B23 was detected in one strain, while the D2 phylogroup was found in 3 isolates. The virulence genes iutA, fimH, and traT were detected in 3, 7, and 3 isolates, respectively. Two types of gene combination were detected: iutA+fimH+traT in 3 isolates and iutA+fimH in one isolate. The isolates recovered in farm 1 showed the same profile of PFGE macro-restriction, while isolates of farm 2 presented unrelated PFGE patterns. We conclude that these avian ESBL-producing E. coli isolates show homo- and heterogenic genetic background and that plasmids harboring ESBL genes could be involved in the dissemination of this resistance phenotype.

PCR-Based Detection of Extended-Spectrum ß-Lactamases (bla CTX-M-1 and bla TEM ) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae Isolated from Pigs in North Eastern India (Mizoram).

Cephalosporins are major antimicrobials used to treat serious infections. However, their effectiveness is being compromised by the emergence of extended-spectrum ß-lactamases (ESBLs). A total of 138 enteric bacteria were isolated from 53 faecal samples of pigs collected from different districts of Mizoram, of which 102 (73.91 %) were Escherichia coli, 26 (18.84 %) were Salmonella spp. and 10 (7.25 %) were Klebsiella pneumoniae. Phenotypic confirmatory test (Double Discs Synergy Test) showed that 8 (5.80 %) E. coli isolates were ESBLs producer. PCR analysis confirmed that out of the eight isolate, 7 (5.07 %) harboured bla CTX-M-1 gene and/or bla TEM gene. Of the eight positive isolates, 7 (5.07 %) and 3 (2.17 %) were found to be positive for bla CTX-M-1 gene and bla TEM gene, respectively, of which 3 (2.17 %) isolates were positive for both the genes. Only 4 (2.90 %) E. coli isolates carried bla CTX-M-1 gene alone. Agarose gel electrophoresis showed that all the isolates were carrying plasmids ranging between 0.9 and ~30 kb. Out of the seven isolates positive for bla CTX-M-1 and/or bla TEM , 2 (1.84 %) isolates were confirmed for bla CTX-M-1 gene in their plasmid. Only one E. coli isolate was found to be positive for both the genes in its plasmid. The resistance plasmid could not be transferred to a recipient by in vitro horizontal gene transfer method.