Occurrence of Pharmaceutically Active Compounds and Potential Ecological Risks in Wastewater from Hospitals and Receiving Waters in Sri Lanka.

The presence of pharmaceutically active compounds (PACs) in the environment and their associated hazards is a major global health concern; however, data on these compounds are scarce in developing nations. In the present study, the existence of 39 non-antimicrobial PACs and six of their metabolites in wastewater from hospitals and adjacent surface waters in Sri Lanka was investigated from 2016 to 2018. The highest amounts of the measured chemicals, including the highest concentrations of atorvastatin (14,620 ng/L) and two metabolites, mefenamic acid (12,120 ng/L) and o-desmethyl tramadol (8700 ng/L), were detected in wastewater from the largest facility. Mefenamic acid, gemfibrozil, losartan, cetirizine, carbamazepine, and phenytoin were detected in all the samples. The removal rates in wastewater treatment were 100% for zolpidem, norsertaline, quetiapine, chlorpromazine, and alprazolam. There was substantial variation in removal rates of PACs among facilities, and the overall data suggest that treatment processes in facilities were ineffective and that some PAC concentrations in the effluents were increased. The estimated risk quotients revealed that 14 PACs detected in water samples could pose low to high ecological risk to various aquatic organisms. Compounds such as ibuprofen, tramadol, and chlorpromazine detected in untreated and treated wastewater at these facilities pose a high risk to several aquatic organisms. Our study provides novel monitoring data for non-antimicrobial PAC abundance and the associated potential ecological risk related to hospitals and urban surface waters in Sri Lanka and further offers valuable information on pre-COVID-19 era PAC distribution in the country. Environ Toxicol Chem 2022;41:298-311. © 2021 SETAC.

The association between antimicrobials and the antimicrobial-resistant phenotypes and resistance genes of Escherichia coli isolated from hospital wastewaters and adjacent surface waters in Sri Lanka.

The presence of antimicrobials, antimicrobial-resistant bacteria (ARB), and the associated antimicrobial resistance genes (ARGs) in the environment is a global health concern. In this study, the concentrations of 25 antimicrobials, the resistance of Escherichia coli (E. coli) strains in response to the selection pressure imposed by 15 antimicrobials, and enrichment of 20 ARGs in E. coli isolated from hospital wastewaters and surface waters were investigated from 2016 to 2018. In hospital wastewaters, clarithromycin was detected at the highest concentration followed by sulfamethoxazole and sulfapyridine. Approximately 80% of the E. coli isolates were resistant, while 14% of the isolates exhibited intermediate resistance against the tested antimicrobial agents. Approximately 61% of the examined isolates were categorized as multidrug-resistant bacteria. The overall abundance of phenotypes that were resistant toward drugs was in the following order: ß-lactams, tetracycline, quinolones, sulfamethoxazole/trimethoprim, aminoglycosides, and chloramphenicol. The data showed that the E. coli isolates frequently harbored blaTEM, blaCTX-M, tetA, qnrS, and sul2. These results indicated that personal care products were significantly associated with the presence of several resistant phenotypes and resistance genes, implying their role in co-association with multidrug resistance. Statistical analysis also indicated a disparity specific to the site, treatment, and year in the data describing the prevalence of ARB and ARGs and their release into downstream waters. This study provides novel insights into the abundance of antimicrobial, ARB and ARGs in Sri Lanka, and could further offer invaluable information that can be integrated into global antimicrobial resistance databases.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3' end of the tRNA genes pheV or pheR The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Transcriptomic analysis of Campylobacter jejuni grown in a medium containing serine as the main energy source.

Campylobacter jejuni is one of the most important causes of food-borne diseases in industrialized countries. Amino acids are an important nutrient source for this pathogen because it lacks enzymes related to glycolysis. However, the metabolic characteristics of C. jejuni grown in a nutrient-restricted medium with specific amino acids have not been fully elucidated. This study shows that C. jejuni NCTC 11168 grows well in a nutrient-restricted medium containing serine, aspartate, glutamate, and proline. Subtracting serine significantly reduced growth, but the removal of the three other amino acids did not, suggesting that serine is a priority among the four amino acids. A transcriptomic analysis of C. jejuni NCTC 11168 grown in a medium with serine as the main energy source was then performed. Serine seemed to be sensed by some chemoreceptors, and C. jejuni reached an adaptation stage with active growth in which the expression of flagellar assembly components was downregulated and the biosyntheses of multiple amino acids and nucleotide sugars were upregulated. These data suggest that C. jejuni NCTC 11168 requires serine as a nutrient.

Phylogenetic Characterization of Salmonella enterica Serovar Typhimurium and Its Monophasic Variant Isolated from Food Animals in Japan Revealed Replacement of Major Epidemic Clones in the Last 4 Decades.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and its monophasic variant (Salmonella 4,[5],12:i:-) are the major causes of gastroenteritis in both humans and animals. Pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis have been used widely as subtyping methods for these pathogens in molecular epidemiological analyses, but the results do not precisely reflect phylogenetic information. In this study, we performed a phylogenetic analysis of these serovars using whole-genome sequencing data and identified nine distinct genotypic clades. Then, we established an allele-specific PCR-based genotyping method detecting a clade-specific single nucleotide polymorphism to rapidly identify the clade of each isolate. Among a total of 815 isolates obtained from cattle in Japan between 1977 and 2017, clades 1, 7, and 9 contained 77% of isolates. Obvious replacement of the dominant clone was observed five times in this period, and clade 9, which mostly contains Salmonella 4,[5],12:i:-, is currently dominant. Among 140 isolates obtained from swine in Japan between 1976 and 2017, clades 3 and 9 contained 64% of isolates. Clade 9 is the latest clone as is the case in cattle isolates. Clade 9 is similar to an epidemic clone from Europe, which is characterized by sequence type 34 (ST34), chromosomal Salmonella genomic island 3, and a composite transposon containing antimicrobial resistance genes. The increased prevalence of clade 9 among food animals in Japan might be a part of the pandemic of the European Salmonella 4,[5],12:i:- clone.

Identification and phase inversion of Salmonella flagellar antigens, using immuno-discs.

Disc immuno-immobilization is a simple method for typing the flagellar phase of Salmonella enterica. We re-examined this method using commercial antisera, which contains the preservative sodium azide. Originally prepared motility agar activates bacterial motility and renders S. enterica resistant to sodium azide, resulting in the formation of immuno-immobilization lines around reactive immuno-discs. Though disc immuno-immobilization serves both serotyping and phase inversion, this method is insufficient for the strains in which phase variation rarely occurs. Here, we devised a novel immuno-disc phase inversion method, and all S. enterica strains tested were identically typed. These methods would drastically simplify the task of S. enterica typing in clinical laboratories.

Characterization of pertussis-like toxin from Salmonella spp. that catalyzes ADP-ribosylation of G proteins.

Salmonella Typhimurium definitive phage type (DT) 104 produces a pertussis-like toxin (ArtAB-DT104), which catalyzes ADP-ribosylation of pertussis toxin sensitive G proteins. However, the prevalence of ArtAB and its toxicity have not been established. We report here that, in addition to DT104, S. Worthington, and S. bongori, produce ArtAB homologs, designated ArtAB-SW and ArtAB-Sb, respectively. We purified and characterized these ArtAB toxins, which comprise a 27-kDa A subunit (ArtA) and 13.8-kDa pentameric B subunits (ArtB). While the sequence of the A subunit, which is ADP-ribosyltransferase, is similar to the A subunit sequences of other ArtABs, the B subunit of ArtAB-Sb is divergent compared to the B subunit sequences of other ArtABs. Intraperitoneal injection of purified ArtABs was fatal in mice; the 50% lethal doses of ArtAB-DT104 and ArtAB-SW were lower than that of ArtAB-Sb, suggesting that ArtB plays an influential role in the toxicity of ArtABs. ArtABs catalyzed ADP-ribosylation of G proteins in RAW 264.7 murine macrophage-like cells, and increased intracellular cyclic AMP levels. ArtAB-DT104 and ArtAB-SW, but not ArtAB-Sb, stimulated insulin secretion in mice; however, unlike Ptx, ArtABs did not induce leukocytosis. This disparity in biological activity may be explained by differences in ADP-ribosylation of target G proteins.

A case study on Salmonella enterica serovar Typhimurium at a dairy farm associated with massive sparrow death.

BACKGROUND: Salmonella enterica Typhimurium (S. Typhimurium) is the most common cause of bovine salmonellosis in Japan and where it is also cause of salmonellosis in wild birds. In 2008, a postpartum cow at a dairy farm developed diarrhea caused by S. Typhimurium. The herd was extensively surveilled for Salmonella sp. and we characterized bacterial isolates from this and other cows to determine the source of infection. RESULTS: Eight isolates of S. Typhimurium from cattle were identified as phage type DT40 and showed a 100 % similarity by pulsed-field gel electrophoresis and the same or similar multiple-locus variable-number tandem-repeat analysis profiles as those of S. Typhimurium isolated from dead sparrows (Passer montanus) collected at Asahikawa in 2006. S. Typhimurium DT40 was considered to be a major cause of high sparrow mortality in Hokkaido in 2005-2006 and 2008-2009, suggesting that DT40 maintained in sparrows was transmitted to cattle. CONCLUSIONS: S. Typhimurium DT40 may be transmitted from sparrows to dairy cattle.

Eosinophilic granuloma with Splendore-Hoeppli material caused by Mannheimia granulomatis in a calf.

A large subcutaneous mass, formed on the left lower jaw of a 4-month-old Japanese Black male calf, was partially excised for histological and bacteriological examinations. Antibiotic treatment resulted in a good prognosis. Bacteria isolated from the excised material were characterized by weak hemolysis and positive reactions for catalase and oxidase, and were 99% identical to Mannheimia granulomatis strains. The presence of the leukotoxin gene product was demonstrated by polymerase chain reaction amplification. Histological examination showed that the excised material was composed of dense fibrous connective tissue with sparsely distributed eosinophilic granulomas or abscesses. These foci frequently contained Splendore-Hoeppli material with rod-shaped Gram-negative bacteria. Except for the absence of lymphangitis and the presence of basophils and mast cells, the histology of this lesion resembled that of lechiguana associated with coinfection of M. granulomatis and Dermatobia hominis. Leukotoxin was demonstrated by immunohistochemistry within Splendore-Hoeppli material and was judged to be responsible for its formation.

Whole-Genome Sequence of CMY-2 ß-Lactamase-Producing Salmonella enterica Serovar Typhimurium Strain L-3553.

Salmonella enterica serovar Typhimurium pulsed-field gel electrophoresis cluster VII has been isolated from cattle populations in Japan since the mid-2000s. Some cluster VII isolates exhibited extended-spectrum cephalosporin resistance defined by the blaCMY-2 gene located in a chromosomal genomic island, GI-VII-6. We determined the whole-genome sequence of strain L-3553 as the reference strain.

Complete nucleotide sequences of virulence-resistance plasmids carried by emerging multidrug-resistant Salmonella enterica Serovar Typhimurium isolated from cattle in Hokkaido, Japan.

In the present study, we have shown that virulence-resistance plasmids from emerging multidrug-resistant isolates of Salmonella enterica serovar Typhimurium were derived from a virulence-associated plasmid, essential for systematic invasiveness of S. Typhimurium in mice (pSLT), through acquisition of a large insert containing a resistance island flanked by IS1294 elements. A bla CMY-2-carrying plasmid from a cefotaxime-resistant isolate comprised a segment of Escherichia coli plasmid pAR060302 and the replication region (IncFIB) of a virulence-resistance plasmid. These results provide insights into the evolution of drug resistance in emerging clones of S. Typhimurium.

Molecular typing of Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates from cattle by multiple-locus variable-number tandem-repeats analysis.

To evaluate the usefulness of multiple-locus variable-number tandem-repeats analysis (MLVA) as a tool for the epidemiological analysis of bovine Salmonellosis, Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates (544 and 18, respectively) obtained from cattle in Hokkaido, Japan, between 1977 and 2009, were characterised by MLVA. MLVA identified 184 profiles versus 121 profiles identified by pulsed-field gel electrophoresis (PFGE). Cluster analysis of the MLVA profiles demonstrated 3 major clusters (A, B, and C) and 3 minor clusters (D, E, and F). Cluster A was associated with PFGE cluster I, which included isolates of definitive phage type 104 (DT104), while cluster C was associated with PFGE cluster VII, which has been disseminating among cattle since 2002. An isolate of serotype Typhimurium belonging to MLVA cluster F, in which 10 serotype 4,5,12:i:- isolates were included, was found to have an MLVA profile closely related to those of serotype 4,5,12:i:- isolates, suggesting that such a strain may be an ancestral candidate for serotype 4,5,12:i:-. Overall, the discriminatory power of MLVA was higher than that of PFGE, and MLVA differentiated between the isolates of the DT104 family, which appeared to be clonal by PFGE. However, this depended on PFGE clusters because PFGE allowed greater discrimination between isolates within PFGE cluster IV and VI than MLVA. The combination of PFGE and MLVA data allowed for improved subtype discrimination and enabled the identification of recently disseminated clones. Hence, MLVA can be used in combination with PFGE to effectively accelerate the molecular epidemiologic investigation of Salmonella.

Change in antimicrobial resistance pattern in Salmonella enterica serovar Typhimurium isolates detected in a beef cattle farm.

Multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates with four different antimicrobial resistance patterns obtained from a beef cattle farm were characterized to determine their clonality. Macrorestriction analysis of genomic DNA revealed that these four isolates are closely related to each other and can be classified as a newly emerged pulsed-field gel electrophoresis type among cattle: cluster VII. Three of the four isolates showed resistance to extended-spectrum cephalosporins (ESCs), and this resistance was mediated by AmpC ß-lactamase encoded by the bla(CMY-2) gene in a 190-kbp IncA/C plasmid. Results of restriction analysis and IncA/C backbone PCR suggest that the three 190-kbp plasmids are identical and that a 70-kbp IncA/C plasmid of the ESC-susceptible isolate is derived from the 190-kbp plasmid by a deletion event. Three isolates harboured a virulence-resistance plasmid (165 or 180 kbp), and restriction analysis revealed that these plasmids were identical or closely related to each other. These results suggest that the four S. Typhimurium cluster VII isolates originate from a common ancestor that probably invaded the farm prior to the salmonellosis outbreak. Antimicrobial resistance patterns may not necessarily reflect the relationships of the isolates.

Plasmid-mediated florfenicol resistance in Mannheimia haemolytica isolated from cattle.

The aim of this study was to analyse a florfenicol-resistant Mannheimia haemolytica isolated from a calf to determine the genetic basis of its florfenicol-resistance. The antimicrobial susceptibility and plasmid content of the isolate were determined. A florfenicol resistant plasmid carrying the floR gene was identified by PCR and transformed into Escherichia coli JM109 and HB101 strains. The plasmid was then mapped and sequenced completely. The isolate was resistant to chloramphenicol, florfenicol, oxytetracycline, kanamycin, dihydrostreptomycin, nalidixic acid, ampicillin, and amoxicillin; it carried a floR plasmid of 7.7kb, designated pMH1405. The mobilisation and replication genes of pMH1405 showed extensive similarity to the 5.1-kb pDN1 plasmid from Dichelobacter nodosus and the 10.8-kb pCCK381 plasmid from Pasteurella multocida. An adjacent 2.4-kb segment was highly homologous to the TnfloR region of the E. coli BN10660 plasmid. A plasmid-mediated floR gene was responsible for florfenicol resistance in the bovine respiratory tract pathogen M. haemolytica. The pMH1405 plasmid is the smallest floR-carrying plasmid reported to date. To the best of our knowledge, this is the first report of a florfenicol-resistant gene in M. haemolytica.

Characterization of Salmonella enterica serovar Typhimurium isolates harboring a chromosomally encoded CMY-2 beta-lactamase gene located on a multidrug resistance genomic island.

Since 2004, extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates have been detected from cattle in the northern major island of Japan, Hokkaido. Resistance to ESCs was found to be mediated by CMY-2 type ß-lactamase among 22 epidemiologically unrelated isolates showing indistinguishable pulsed-field gel electrophoresis patterns. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that the CMY-2 ß-lactamase gene (bla(CMY-2)) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S. Typhimurium isolate L-3553 indicated that bla(CMY-2) was located on a unique 125-kb genomic island, GI-VII-6, which consists of 140 open reading frames. Pairwise alignment of GI-VII-6 and Escherichia coli plasmid pAR060302 (size, 167 kb) revealed that a large proportion of GI-VII-6 (99%) shows a high sequence similarity (>99%) with pAR060302. GI-VII-6 contains 11 antimicrobial resistance genes including sul1, qacEΔ1, aadA2, and dfrA12 in the aadA2 region; sugE1 and bla(CMY-2) in the bla(CMY-2) region; and sul2, strA, strB, tet(A), and floR in the floR region. Two directly repeated IS26 copies were present at both ends of GI-VII-6. Junction regions of GI-VII-6 were flanked by an 8-bp direct repeat, indicating that GI-VII-6 was acquired by transposition involving IS26 transposase. PCR scanning revealed that the overall structure of GI-VII-6 was almost identical in the 22 isolates. Phylogenetic analysis suggested that S. Typhimurium isolates harboring GI-VII-6 belong to a different genomic lineage than other whole-genome-sequenced S. Typhimurium strains. These data indicate that a particular clone of S. Typhimurium harboring GI-VII-6 has spread among the cattle population in Hokkaido, Japan.

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone.

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.