Molecular Detection of Ampicillin Resistant Genes in E. coli Isolates from ‎Dogs in India

The most common cause of urinary tract infection in dogs is uropathogenic Escherichia coli (UPEC). This condition often presents with vaginal discharge, dribbling of urine, straining or vocalization while urinating due to pain. Furthermore, the following signs are also noticeable: hematuria, lethargy, proteinuria, dysuria, cystitis, and oliguria. The aim of this research was to investigate the genes of ampicillin resistance in E. coli isolates from dogs with urinary tract infections. Out of 103 urine samples cultured (Blood agar, MacConkey’s lactose agar and Eosin methylene blue agar), 24.3% were positive for uropathogenic Escherichia coli. The positive isolates were further subjected to antimicrobial sensitivity test and PCR analysis. All the uropathogenic Escherichia coli isolates were resistant to ampicillin while 96% were resistant to Cloxacillin and Oxytetracycline. Susceptibility to Meropenem, Gentamicin and Amikacin were 64 %, 44 % and 40% respectively. All the 25 strains of the E. coli were identified to be resistant to two or more antibiotics. The PCR result showed the presence of blaAMPC in all the samples and 60 % had blaTEM genes responsible for ampicillin resistance. However, none of the isolates were positive for the blaSHV gene.The presence of the blaAMPC and blaTEM genes in the dogs studied resulted in ampicillin resistance, with blaAMPC being the most commonly detected ampicillin gene in Escherichia coli in the study area. Meropenem was also found to be a good choice for treating uropathogenic E. coli infection in dogs.

An update on ampicillin resistance and β-lactamase genes of Bacteroides spp.

Introduction. There are several β-lactamase genes described for Bacteroide s strains, of which cepA and cfiA are specific for Bacteroides fragilis and define two genetic divisions. The expression and phenotypic effects of these genes are usually regulated by insertional activation. Hypotheses/Gap Statement. Information is lacking about how cepA is regulated for most of the B. fragilis strains and whether there could be a genetic element for it. Aim. We aimed to investigate the molecular background of ampicillin (and other β-lactam) resistance among Bacteroides strains as mediated mainly by cepA and also to find a genetic element for it as known for cfiA. Methodology. Various PCR methods were used for β-lactamase-resistance gene and insertion sequence (IS) element detection in 42 Bacteroides strains. β-Lactamase activity measurements and antimicrobial-susceptibility testing using agar dilution were also applied. Further molecular experiments involved sequencing, gene targeting, Southern blotting and bioinformatic analyses. Results. We found that high antibiotic resistance and β-lactamase levels are brought about by insertional activation of the cepA gene or by similar or dissimilar activation of cfxA or cfiA, or by the newly described pbbA genes. Non-activated cepA genes produced low levels of specific β-lactamase activities that did not correlate with ampicillin resistance. We found a genetic element for cepA and another region close to it that are characteristic for division I B. fragilis strains, which are replaced by other sequences in division II B. fragilis strains. Conclusion. cepA usually is not activated by IS elements and usually produces low β-lactamase activities that do not correlate with the ampicillin MICs; therefore, it probably involves some non-β-lactamase-mediated resistance mechanism(s). pbpA is a newly described, effective β-lactamase gene that is located on a plasmid, and cepA resides on a well-defined chromosomal segment that is mutually replaced in division II B. fragilis strains. This latter finding demonstrates the genetic dichotomy of cepA–cfiA in B. fragilis and requires further investigation.

Genome-based studies indicate that the Enterococcus faecium Clade B strains belong to Enterococcus lactis species and lack of the hospital infection associated markers

Enterococcus lactis and the heterotypic synonym Enterococcus xinjiangensis from dairy origin have recently been identified as a novel species based on 16S rRNA gene sequence analysis. Enterococcus faecium type strain NCTC 7171T was used as the reference genome for determining E. lactis and E. faecium to be separate species. However, this taxonomic classification did not consider the diverse lineages of E. faecium , and the double nature of hospital-associated (clade A) and community-associated (clade B) isolates. Here, we investigated the taxonomic relationship among isolates of E. faecium of different origins and E. lactis , using a genome-based approach. Additional to 16S rRNA gene sequence analysis, we estimated the relatedness among strains and species using phylogenomics based on the core pangenome, multilocus sequence typing, the average nucleotide identity and digital DNA–DNA hybridization. Moreover, following the available safety assessment schemes, we evaluated the virulence profile and the ampicillin resistance of E. lactis and E. faecium clade B strains. Our results confirmed the genetic and evolutionary differences between clade A and the intertwined clade B and E. lactis group. We also confirmed the absence in these strains of virulence gene markers IS16, hylEfm and esp and the lack of the PBP5 allelic profile associated with ampicillin resistance. Taken together, our findings support the reassignment of the strains of E. faecium clade B as E. lactis .

Haemophilus influenzae Vulvovaginitis in Prepubertal Girls: A 4-Year Study on a Tertiary Children’s Hospital in China

Background: Vulvovaginitis is a common infection in prepubertal girls, which is partly caused by bacterial infection. According to the literature, Haemophilus influenzae is one of the most common bacterial causes of vulvovaginitis in children. However, few studies with large sample sizes have delved into this issue. Objectives: To determine the prevalence of Haemophilus influenzae vulvovaginitis in prepubertal girls and detect the antimicrobial resistance of H. influenzae strains isolated from vulval specimens. Methods: The isolates of H. influenzae from the vulval swabs of prepubertal girls with vulvovaginitis were received from The Children’s Hospital, Zhejiang University School of Medicine, during 2016 - 2019. The vulval specimens were inoculated on Haemophilus selective chocolate agar, and antimicrobial susceptibility tests were performed by the disk diffusion method. Moreover, β-lactamase was detected using Cefinase disc. Results: In this study, 4142 vulval specimens were received during four years, of which 649 H. influenzae isolates had been isolated from 642 girls aged 6 months-13 years, with a median of 5 years. The peaks of isolates were observed from April to July in the vulval isolates. In general, the ampicillin resistance rate was 39.1% (250/640), 33.2% of strains (211/636) were β-lactamase-positive isolates, and 6.6% strains (42/635) were β-lactamase-negative ampicillin-resistant" (BLNAR)) isolates. The resistance rates of H. influenzae isolates to amoxycillin-clavulanic acid, ampicillin-sulbactam, cefuroxime, ceftriaxone, cefotaxime, meropenem, levofloxacin, sulfamethoxazole-trimethoprim, azithromycin, and chloramphenicol were 26.4%, 21.8%, 24.8%, 1.7%, 1.0%, 0.2%, 0%, 47.7%, 10.2%, and 1.1%, respectively. MDR was noticed in 41 persons (6.4%) out of the 642 H. influenzae isolates, with the most prevalent MDR phenotype of ampicillin-sulfamethoxazole-trimethoprim-azithromycin resistance. Conclusions: Clinicians should noticed that H. influenzae is a common bacterial cause of vulvovaginitis in children, and laboratories should routinely cover Haemophilus culture media for vulval specimens. The Ampicillin resistance of H. influenzae should also be considered in clinical management.

Copy-out-paste-in transposition of a Tn6283-like integrative element assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis

The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multi-drug resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor strain and multidrug-resistant transconjugants. Sequence analysis revealed a conjugative plasmid of the MOBH family in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb integrative element, designated Tn 6945, harboring a beta-lactamase gene that conferred ampicillin resistance to the host cell. Use of a recA mutant E. coli strain as the recipient yielded a transconjugant showing ampicillin resistance but not multidrug resistance, suggesting the involvement of homologous recombination in plasmid integration into the chromosome. Polymerase chain reaction experiments revealed that Tn 6945 generates a circular copy without generating an empty donor site, suggesting that it moves via a copy-out-paste-in mode, as previously reported for Tn 6283. Transposition of the integrative element into multiple loci in the recipient chromosome increased the resistance level of the transconjugants. Overall, these results suggest that Tn 6283-like copy-out integrative elements and conjugative plasmids additively spread AMR genes among marine bacteria and contribute to the emergence of isolates with high-level resistance through amplification of AMR genes.

Prevalence and Antimicrobial Resistance of Salmonella Isolated From Dead-in-Shell Chicken Embryos in Shandong, China

The present study was designed to explore the Salmonella prevalence and antimicrobial resistance characteristics in the context of chick mortality at hatching in China. Between December 2015 and August 2017, 1,288 dead-in-shell chicken embryos were collected from four breeder chicken hatcheries in Tai'an, Rizhao, Jining, and Heze, China. Salmonella isolates were successfully recovered from 6.7% of these embryos (86/1,288) and were evaluated for serotype, antimicrobial susceptibility, Class 1 integron prevalence, antimicrobial resistance gene expression, and multilocus sequence typing (MLST). Salmonella Thompson (37.2%), and Salmonella Infantis (32.6%) were the two most prevalent isolates in these chicken embryos, and 66.3% of isolates exhibited robust ampicillin resistance, while 55.8% of isolates exhibited multi-drug resistance (MDR). The majority of isolates harbored the blaTEM gene (74.4%), with the qnrS gene also being highly prevalent (50.0%). In contrast, just 27.9% of these isolates carried Class 1 integrons. These 86 isolates were separated into four sequence types (STs), whereby ST26 (32.2%) was the most prevalent. Overall, these results suggested that Salmonella infections may be an important cause of chicken embryo mortality in China, and that efforts to support the appropriate use of antibiotics in managing poultry populations are essential.

Genomic diversity and antimicrobial resistance of Haemophilus colonising the airways of young children with cystic fibrosis

Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae (Hi) and Haemophilus parainfluenzae (Hpi) are routinely isolated from the lungs of children with CF, however little is known about the frequency and characteristics of Haemophilus colonisation in this context. Here, we describe detection, antimicrobial resistance (AMR) and genome sequencing of Hi/Hpi isolated from sputum, cough swab, and bronchoalveolar lavage samples regularly collected from 147 participants aged ≤12 years enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program. The frequency of colonisation per visit was 4.6% for Hi and 32.1% for Hpi, 80.3% of participants had Hi and/or Hpi detected on at least one visit, and using genomic data we estimate 15.6% of participants had persistent colonisation with the same strain for at least two consecutive visits. Colonising strains were genetically highly diverse and AMR was common, with 52% of Hi and 82% of Hpi displaying resistance to at least one drug. The genetic basis for AMR could be identified in most cases; novel determinants include a new plasmid encoding blaTEM-1 (ampicillin resistance), a new inhibitor-resistant blaTEM allele (augmentin resistance), and previously unreported mutations in chromosomally-encoded genes (pbp3, ampicillin resistance; folA and folP, co-trimoxazole resistance; rpoB, rifampicin resistance). Acquired AMR genes were significantly more common in Hpi than Hi (51% vs 21%, p=0.0107) and were mostly associated with the ICEHin mobile element carrying blaTEM-1, resulting in higher rates of ampicillin resistance in Hpi (73% vs 30%, p=0.0004). The genome data identified six potential instances of Haemophilus transmission between participants, three of which involved participant pairs who attended the clinic on the same day. The high prevalence of Haemophilus colonisation and high burden of antimicrobial use in children with CF likely provides a reservoir for emergence and spread of AMR as well as a source of infections.

Invasive Haemophilus influenzae Infections in Germany After the Introduction of Routine Childhood Immunization, 2001–2016

Background Haemophilus influenzae (Hi) serotype b (Hib) vaccination was introduced in Germany in 1990. This study presents a comprehensive overview on the burden of invasive Hi infections for 2001–2016, including serotype distribution and ampicillin resistance. Methods Nationwide data from statutory disease surveillance (2001–2016) were linked with laboratory surveillance data (2009–2016). Besides descriptive epidemiology, statistical analyses included multiple imputation to estimate secular trends. Results In 2001–2016, 4044 invasive Hi infections were reported. The mean incidence was 3.0 per million inhabitants, higher in males (3.2 vs 2.9 in females) and in the age groups <1 year (15.2) and ≥80 years (15.5). Nontypeable Hi (NTHi) caused 81% (n = 1545) of cases in 2009–2016. Of capsulated cases, 69% were serotype f and 17% serotype b. Of Hib cases eligible for vaccination, 10% (3/29) were fully vaccinated. For 2009–2016, significant increasing trends were observed for NTHi and Hif infections in the age groups <5 years and ≥60 years and for ampicillin resistance in NTHi. Conclusions This is one of the most comprehensive Hi data analyses since the introduction of Hib vaccines. NTHi and Hif cause an increasing disease burden among elderly patients and infants. Ampicillin resistance in NTHi must be considered in the treatment of invasive Hi infections.

Australian Group on Antimicrobial Resistance (AGAR) Australian Enterococcal Sepsis Outcome Programme (AESOP) Annual Report 2019

From 1 January to 31 December 2019, thirty-nine institutions around Australia participated in the Australian Enterococcal Sepsis Outcome Programme (AESOP). The aim of AESOP 2019 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the E. faecium isolates. Of the 1,361 unique episodes of bacteraemia investigated, 95.2% were caused by either E. faecalis (51.4%) or E. faecium (43.8%). Ampicillin resistance was not detected in E. faecalis but was detected in 91.1% of E. faecium. Vancomycin non-susceptibility was detected in 0.1% of E. faecalis and in 41.8% of E. faecium. Overall, 45.4% of E. faecium harboured vanA and/or vanB genes. For the vanA/vanB positive E. faecium isolates, 49.1% harboured vanA genes only and 50.6% vanB genes; 0.3% harboured both vanA and vanB genes. The percentage of E. faecium bacteraemia isolates resistant to vancomycin in Australia is substantially higher than that seen in most European countries. E. faecium consisted of 78 multilocus sequence types (STs), of which 75.0% of isolates were classified into six major STs containing ten or more isolates. All major STs belong to clonal cluster (CC) 17, a major hospital-adapted polyclonal E. faecium cluster. The predominant STs (ST1424, ST17, ST796, ST80, ST1421, and ST78) were found across most regions of Australia. The most prevalent clone was ST1424, which was identified in all regions except the Northern Territory and Western Australia. Overall, 51.4% of isolates belonging to the six predominant STs harboured vanA or vanB genes. In 2019, AESOP has shown that enterococcal bacteraemias in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin-resistant vanA or vanB E. faecium which have limited treatment options.