Identification and characterization of multidrug-resistant ESBL-producing Salmonella enterica serovars Kentucky and Typhimurium isolated in Tunisia CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing ß-lactamase of Salmonella.

AIMS: Molecular characterization of extended-spectrum ß-lactamases (ESBLs) among Salmonella Kentucky and Typhimurium isolates: partial sequence analysis of the types of ß-lactamases found in these isolates, clonality, resistance and supposed emergence of ESBL-producing strains. METHODS AND RESULTS: A retrospective study surveyed the ESBLs occurring in a total of 1404 Salmonella Kentucky and Typhimurium isolates collected over a 5-year period in Tunisia. Antimicrobial susceptibility tests, ESBL phenotype determination (double-disc synergy) were performed. Polymerase chain reaction assays were used for the detection of ß-lactamase genes (blaTEM , blaSHV , blaOXA-1 and blaCTX-M ), class 1 and class 2 integrases (intI1 and intI2) and the 3' conserved segment (3'-CS) of class 1 integron (qacEΔ1+sul1). Sequencing of amplicons of ß-lactamase genes was performed. Percentage of 9.8 of the isolates (S. Kentucky = 117, S. Typhimurium = 20) were either resistant to penicillin and had decreased susceptibility to cefotaxime or had a positive double-disc synergy test result. Polymerase chain reaction detected that these isolates harboured one or more ß-lactamase genes (blaTEM , blaSHV , blaOXA-1 or blaCTX-M ). TEM-1, TEM-34, CTX-M15, CTX-M9 and CTX-M61 type ESBLs were identified through sequencing. The novel Salmonella cefotaxime-hydrolysing ß-lactamase, CTX-M61/TEM-34, detected in this study showed the emergence of new CTX-M-type ESBLs in Tunisia. There were found 33 different multidrug resistance (MDR) patterns. CONCLUSION: These findings highlighted the proliferation of ESBLs and MDR in Salmonella Kentucky and Typhimurium isolates from numerous regions and sources in Tunisia, indicating an emerging public health concern. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing ß-lactamase of Salmonella had been detected.

Short communication: Diversity of staphylococci isolated from sheep mastitis in northern Algeria.

Mastitis in ruminants is an important disease with major effects on both the economy and animal welfare. It is caused by major pathogens such as Staphylococcus aureus and minor pathogens such as coagulase-negative staphylococci. The objective of this study was to identify and characterize staphylococci as a cause of sheep mastitis in Algeria. In this study, 123 milk samples were collected directly from the udder of sheep suffering from clinical mastitis in 2 provinces in Algeria. Recovered isolates were identified using MALDI-TOF mass spectrometry. Virulence-associated and antimicrobial resistance genes as well as clonal complexes (CC) of S. aureus were determined using microarray-based analysis. A total of 45 staphylococci isolates were cultivated from sheep milk samples, and 28 S. aureus were identified as methicillin susceptible (62.2%). Seventeen other Staphylococcus isolates of different species were identified using MALDI-TOF mass spectrometry. Subsequent microarray analysis typed the methicillin-susceptible S. aureus to 6 CC: CC8-MSSA, CC97-MSSA, CC130/521-MSSA, CC479-MSSA, CC522-MSSA, and CC705-MSSA. The accessory gene regulator agrIII and the ruminant leukocidin genes lukF-P83 and lukM were found in all isolates of CC130/521, CC479, CC522, and CC705. The toxic shock syndrome toxin gene tst1 was detected exclusively in CC130/521. Additionally, virulence-associated genes (sea, sed, sak, hld, hlgA, edinB, and others) were detected. The presence of antibiotic resistance genes [blaZ, erm(B), and tet(K)] was detected in small numbers of staphylococci. Staphylococci possessing these genes are considered potential hazards for farm animals, farmers, and consumers. Data concerning the prevalence and diversity of staphylococci causing mastitis in sheep from Algeria are lacking. Presented results on different aspects about staphylococci in Algerian sheep populations should at least partially close that gap. However, further extensive studies covering more geographical regions are needed to assess the epidemiological risk.

Identification, differentiation and antibiotic susceptibility of Gallibacterium isolates from diseased poultry.

BACKGROUND: Gallibacterium anatis is an opportunistic pathogen of intensively reared poultry causing oophoritis, salpingitis, peritonitis and enteritis. Gallibacterium anatis infection often remains undiagnosed. Recently multi-drug resistant isolates have been described. METHODS: A newly developed PCR restriction fragment length polymorphism assay targeting the 16S rRNA gene was used to identify and differentiate Gallibacterium isolates from chicken, turkey and partridge samples originating from 18 different geographical locations in Thuringia, Germany. Antimicrobial susceptibility to 19 compounds of different classes was assessed. RESULTS: Nineteen Gallibacterium isolates were investigated. In 9 birds (47.4%) Gallibacterium species were isolated exclusively while in 10 birds (52.6%) other bacterial or viral agents could be detected in addition. In one chicken a mixed infection of Gallibacterium anatis and Gallibacterium genomospecies was identified. All isolates were susceptible to apramycin, florfenicol and neomycin and resistant to clindamycin, sulfathiazole and penicillin. Resistance to sulfamethoxim, spectinomycin, tylosin and oxytetracycline was observed in 93.3%, 93.3%, 86.7% and 80.0% of the field strains, respectively. CONCLUSIONS: The PCR-RFLP assay allows specific detection and differentiation of Gallibacterium spp. from poultry. Antimicrobial resistance of Gallibacterium spp. is highly significant in Thuringian field isolates.

Human Brucellosis in Febrile Patients Seeking Treatment at Remote Hospitals, Northeastern Kenya, 2014-2015.

During 2014-2015, patients in northeastern Kenya were assessed for brucellosis and characteristics that might help clinicians identify brucellosis. Among 146 confirmed brucellosis patients, 29 (20%) had negative serologic tests. No clinical feature was a good indicator of infection, which was associated with animal contact and drinking raw milk.

Brucellosis in pregnant women from Pakistan: an observational study.

BACKGROUND: Brucella species occasionally cause spontaneous human abortion. Brucella can be transmitted commonly through the ingestion of raw milk or milk products. The objective of this study was to determine the sero-prevalence of and to identify potential risk factors for brucellosis in pregnant women from Rawalpindi, Pakistan. METHODS: We conducted a cross-sectional study at the Gynecology Outdoor Patient department of the Benazir Bhutto Hospital, Rawalpindi, Pakistan from March to June 2013. Data related to potential risk factors and clinical history was collected by individual interviews on the blood sampling day. The 429 serum samples collected were initially screened by Rose Bengal Plate Agglutination test for the detection of Brucella antibodies. We applied standard descriptive statistics and logistic regression analyses. RESULTS: Twenty five (5.8 %; 95 % confidence interval (CI): 3.8 % -8.5 %) serum samples were found to be seropositive. Brucellosis-related clinical symptoms were recorded in various seropositive cases. Animal contact, raw milk consumption, having an abortion history and the experience of an intrauterine fetal death were associated with seropositivity for brucellosis in univariate analyses (all p <0.05). In multiple logistic regression models only the contact with animals remained as independent and robust risk factor (odds ratio 5.21; 95 % CI: 1.88-13.75; p = 0.001) for seropositivity. CONCLUSION: Brucellosis is a serious threat for pregnant women and their unborn children in Pakistan. Pregnant women having brucellosis-related symptoms or previous history of abortions, miscarriages, intrauterine fetal death and other brucellosis-related manifestations should be screened for brucellosis - especially those exposed to animals given the increased risk - and medication should be administered according to state of the art.

Serological, molecular detection and potential risk factors associated with camel brucellosis in Pakistan.

Brucellosis is one of the most important zoonoses in developing countries and was considered the most widespread zoonosis in the world. Brucellosis was reported in camels and has been reported from all camel-keeping countries.The present study was performed in three districts (Jhang, Chiniot, and Bhakkar) of Punjab province of Pakistan. A total of 200 camel (Camelus bactrianus) sera were collected using random and multistage cluster sampling from different areas. Fifty samples were collected from one organized governmental farm. One hundred fifty samples were collected randomly from nomadic/pastoral production systems. All sera were tested with Rose Bengal plate agglutination test (RBPT) and confirmed by ELISA. Genomic DNA was extracted from all serum samples and tested by real-time PCR. Various potential risk factors (season, rearing with other animals, and abortion or orchitis history) recorded through questionnaires were statistically analyzed by Chi-square test.In total, 5 % of investigated sera were positive by RBPT. Only 2 % of the camel sera were CELISA positive. Brucella abortus DNA was detected in 1.5 % of the investigated animals. Season, rearing of camels with other ruminants, abortion, and orchitis history were found to be statistically significant (p < 0.05) disease for determinants.Camel brucellosis is a zoonotic disease in the Pakistani Punjab with various risk factors maintaining and perpetuating its spread. Therefore, there is a need for implementing control measures and raising public health awareness in prevention of brucellosis in Pakistan.

Serological and molecular investigation for brucellosis in swine in selected districts of Uganda.

Brucellosis is a notifiable zoonotic disease affecting livestock, humans, and wildlife in Uganda. Pigs can be infected with human pathogenic Brucella suis biovars 1 and 3 and can be a significant source of brucellosis for humans. Uganda has a rapidly growing pig population, and the pork consumption per capita is the highest in East Africa. The objective of this work was to determine the seroprevalence of brucellosis in Ugandan pigs. A cross-sectional serosurvey of pigs was conducted in three of the major pig-keeping districts in Uganda (Masaka (n = 381 samples), Mukono (n = 398), and Kamuli (n = 414)). In addition, pigs originating from these districts were sampled in the major pig abattoir in Kampala (n = 472). In total, 1665 serum samples were investigated by serological and molecular tests. Only three putative brucellosis-positive samples were detected serologically using indirect ELISA. These sera were found negative for Brucella antibodies by CFT; however, two had antibodies against Yersinia enterocolitica as determined by SAT. Presence of antibodies against Yersiniae was confirmed by Y. enterocolitica antibody-specific ELISA. The two Yersiniae ELISA-positive samples were brucellosis negative using real-time PCR. We tested additional 142 sera from the 1665 samples with real-time PCR. All tested negative. Under this type of production system, we expect a maximum B. suis prevalence of less than 1 % at 95 % confidence level, and therefore, the risk of acquiring brucellosis from the pigs or their products is negligible. However, pigs may harbor the zoonotic Y. enterocolitica. This is the first study to investigate the occurrence of brucellosis in pigs in Uganda and the first study to report Y. enterocolitica antibodies in swine in Uganda.

Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli recovered from organic turkey farms in Germany.

The popularity of food produced from animals kept under an organic regimen has increased in recent years. In Germany, turkey meat consumption has increased. Despite several studies assessing the susceptibility of campylobacters to various antibiotics in poultry, no sufficient data exists regarding the antimicrobial resistance of campylobacters in organic-reared turkeys. This study provides information about antibiotic resistance in Campylobacter isolated from turkeys reared on organic farms in Germany. Ninety-six Campylobacter strains (41 C. jejuni and 55 C. coli) were isolated from different free-range turkey flocks. In vitro antimicrobial sensitivity testing was done using a broth microdilution test, and the presence of resistance genes to antibiotics (ciprofloxacin, tetracycline) was investigated. All Campylobacter isolates from organic turkeys (n = 96) were phenotypically sensitive to gentamicin, erythromycin, streptomycin, and chloramphenicol. In this study, the antibiotic susceptibilities of C. jejuni to ciprofloxacin, tetracycline, and naladixic acid were 56.0%, 51.3%, and 56.0%, respectively. In contrast, 44.0%, 73.0%, and 74.6% of C. coli isolates were resistant to tetracycline, ciprofloxacin, and nalidixic acid, respectively. Replacement of the Thr-86→Ile in the gyrA gene, and the presence of the tet(O) gene were the mainly identified resistance mechanisms against fluoroquinolones and tetracycline, respectively.These results also reinforce the need to develop strategies and implement specific control procedures to reduce the development of antimicrobial resistance.

Isolation of the highly pathogenic and zoonotic agent Burkholderia pseudomallei from a pet green Iguana in Prague, Czech Republic.

BACKGROUND: Melioidosis caused by Burkholderia (B.) pseudomallei is an endemic zoonotic disease mainly reported from northern Australia and Southeast Asia. In Europe, cases of human melioidosis have been reported only from patients travelling to endemic regions. Besides humans, B. pseudomallei has a very broad host range in domestic and wild animals. There are some reports about importation of B. pseudomallei-infected animals from endemic areas into Europe. The present report describes the first case of B. pseudomallei infection of a pet iguana in Europe. CASE PRESENTATION: In a 5-year-old pet Iguana iguana living in a private household in Prague, Czech Republic, B. pseudomallei was isolated from pus of an abscess. The isolate VB976100 was identified by Vitek®2, MALDI-TOF mass spectrometry and polymerase chain reaction as B. pseudomallei. The molecular typing resulted in multi-locus sequence type 436 hitherto, which has been found only once worldwide in a B. pseudomallei strain isolated in the USA and originating from Guatemala. The identification as internal transcribed spacer type G indicates a close relatedness to strains mainly isolated in the Western Hemisphere. These findings support the hypothesis that the iguana became infected in this region or in a breeding facility through contact to other infected animals. CONCLUSIONS: The present case highlights the risk of importation of the highly pathogenic and zoonotic B. pseudomallei into non-endemic regions through animal trade. Therefore, veterinarians treating animals from these areas and physicians examining patients owning such animals should include melioidosis in differential diagnosis whenever specific symptoms appear. Furthermore, veterinary authorities responsible for supervision of traders and pet shops should be aware of this risk of zoonotic transmission.

Whole-genome sequencing of Streptococcus uberis isolated from cows with mastitis in Thuringia.

Introduction. Streptococcus uberis is a common cause of mastitis in cattle, leading to significant economic losses. The widespread use of antimicrobials has contributed to the emergence of resistance, which poses a severe challenge in controlling S. uberis infection.Aim. The objective of this study was to gain insights into the antimicrobial resistance (AMR) and epidemiological typing of S. uberis isolated from milk collected from bovine mastitis on dairy farms in Thuringia.Methodology. In this study, 84 S. uberis isolates were obtained from cattle with clinical mastitis in Thuringia, their phenotypic and genotypic AMR were analyzed and their phylogenetic relationship was explored using whole-genome sequencing.Results. Genetically heterogeneous strains were found on the farms, but clusters of highly similar strains also circulated within the same farms. All isolates were sensitive to ampicillin, penicillin, ceftiofur, and vancomycin. However, 42.9%, 42.9%, 22.6%, 19.0%, and 13.0% were resistant to tetracycline, doxycycline, clindamycin, pirlimycin, and erythromycin, respectively. Thirty-nine strains were phenotypically resistant to two or more tested antibiotics. We identified a plasmid associated with macrolide and lincosamide resistance in 12% of the strains.Conclusion. The emergence of S. uberis strains resistant to multiple antibiotics highlights the importance of S. uberis surveillance and the prudent use of antimicrobials.

Seroprevalence and molecular detection of brucellosis among Pakistani women with spontaneous abortion.

Background: Human brucellosis is a neglected disease transmitted to humans from animals such as cattle, goats, dogs, and swine. The causative agents are bacteria of the genus Brucella, intracellular pathogens usually confined to the reproductive organs of their animal hosts causing sterility and abortions. The objective of the study was to determine the seroprevalence of brucellosis among women with spontaneous abortions (SAW) and compare this seroprevalence with that of healthy pregnant women (HPW). Methods: The case-control study was designed to determine the seroprevalence and molecular detection of brucellosis in women who suffered from spontaneous abortion and healthy pregnant women of the Haripur District of Pakistan. A total of 770 blood samples (n = 385 for each group) were collected from 9 public and 11 private hospitals in Haripur District from December 2021-March 2023. Data on demographic features, epidemiological variables, and risk factors were collected from each participant by structured questionnaires. Initial screening for brucellosis was performed by Rose Bengal Plate Test followed by qRT-PCR for molecular detection of the genus-specific BCSP-31 gene of Brucella. Results: The study showed that anti-Brucella antibodies were more found in SAW 23.63% (91/385) than in HPW 1.29% (5/385). Brucella specific DNA was amplified in 89.01% (81/91) seropositive samples of SAW. Demographic features and risk factors such as age, urbanicity, socioeconomic status, education, occupation, and animal contact were found significantly associated with brucellosis (p ≤ 0.05). Consumption of unpasteurized raw milk (OR = 18.28, 95%CI: 8.16-40.94) was found highly concomitant with seroprevalence. Conclusion: This study reports the first evidence of involvement of brucellosis in spontaneous abortions in women of Pakistan. The study can be used to develop strategies for risk management during pregnancy, to raise awareness for brucellosis, and develop control programs.

The History of Bovine Genital Campylobacteriosis in the Face of Political Turmoil and Structural Change in Cattle Farming in Germany.

Contagious bovine genital campylobacteriosis (BGC), also known as bovine venereal campylobacteriosis, is a disease relevant to international trade listed by the World Organization for Animal Health (WOAH). It is caused by Campylobacter fetus subsp. venerealis (Cfv), one of three subspecies of Campylobacter fetus. Bulls are the reservoir but BGC may also be spread by artificial insemination (AI). BGC is characterized by severe reproductive losses such as infertility, early embryonic death and abortion with considerable economic losses. This significant economic impact has prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. While there are commercial and autologous vaccines available, scientific evidence for the effectiveness of vaccination is still lacking. In Germany, BCG was already found to be endemic in the 1920s, shortly after the agent and the disease had been described for the first time. It can be assumed that BCG had already circulated uncontrolled for a long time in the predecessor states of Germany, influenced only by the political situation and trading networks of the time. After WW II, BCG was eradicated in the German Democratic Republic due to industrialized cattle production based on AI but it was still endemic at low levels in the Federal Republic of Germany with its diverse cattle production. There has been a steady decline in BGC incidence in re-unified Germany over the past 28 years. A single genetic Cfv lineage was identified which probably emerged in the 19th century and diversified over time. Interestingly, no recurrent cross-border introduction became evident. This review gives insight into the history of bovine genital campylobacteriosis considering the structural change in cattle farming in Germany and reflecting on the political background of the time.

Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum in Poultry: Review of Pathogenesis, Antibiotic Resistance, Diagnosis and Control in the Genomic Era.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) has two distinct biovars, Pullorum and Gallinarum. They are bacterial pathogens that exhibit host specificity for poultry and aquatic birds, causing severe systemic diseases known as fowl typhoid (FT) and Pullorum disease (PD), respectively. The virulence mechanisms of biovars Gallinarum and Pullorum are multifactorial, involving a variety of genes and pathways that contribute to their pathogenicity. In addition, these serovars have developed resistance to various antimicrobial agents, leading to the emergence of multidrug-resistant strains. Due to their economic and public health significance, rapid and accurate diagnosis is crucial for effective control and prevention of these diseases. Conventional methods, such as bacterial culture and serological tests, have been used for screening and diagnosis. However, molecular-based methods are becoming increasingly important due to their rapidity, high sensitivity, and specificity, opening new horizons for the development of innovative approaches to control FT and PD. The aim of this review is to highlight the current state of knowledge on biovars Gallinarum and Pullorum, emphasizing the importance of continued research into their pathogenesis, drug resistance and diagnosis to better understand and control these pathogens in poultry farms.

Whole genome sequence-based analysis of Staphylococcus aureus isolated from bovine mastitis in Thuringia, Germany.

Background: Bovine mastitis is a common disease of dairy cattle causing major economic losses due to reduced yield and poor quality of milk worldwide. The current investigation aimed to gain insight into the genetic diversity, antimicrobial resistance profiles and virulence associated factors of Staphylococcus (S.) aureus isolated from clinical bovine mastitis in dairy farms in Thuringia, Germany. Methods: Forty Staphylococcus aureus isolates collected from clinical bovine mastitis cases from 17 Thuringian dairy farms were phenotyped and genetically characterized using whole genome sequencing. Results: Out of 40 S. aureus, 30 (75%) were confirmed as methicillin resistant isolates. The isolates showed elevated antimicrobial resistance against penicillin, tetracycline and oxacillin, i.e., 77.5, 77.5, and 75%, respectively. Lower resistance rates were found against moxifloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, i.e., 35, 35, 30, and 22.5%, respectively. While resistance against clindamycin and erythromycin was rarely found (5 and 2.5%, respectively). All isolates were susceptible to linezolid, teicoplanin, vancomycin, tigecycline, fosfomycin, fusidic acid and rifampicin. These isolates were further allocated into five different sequence types: ST398 (n = 31), ST1074 (n = 4), ST504 (n = 3), ST582 (CC15) (n = 1) and ST479 (n = 1). These isolates were also assigned to seven clusters with up to 100 SNP which has facilitated geographical mapping and epidemiological distribution in Thuringia. Strains belonging to ST398 were classified into clusters 1, 2, 3, 4 and 7. The isolates of ST504 were of cluster 5, those of ST1074 were belonging to cluster 6. Resistance genes blaZ, blaI and blaR associated with penicillin resistance were found in 32 (80%) strains, all except one were belonging to ST398. Methicillin resistance associated mecA was identified in 30 (96.8%) isolates of ST398. All tetracycline and erythromycin resistant isolates were of ST398, and all harbored both tetM and ermA. About 90.3% of tetracycline resistant isolates assigned to ST398 were also carrying tetK gene. The point mutations parC_S80F, gyrA_S84L and parC_S80Y in gyrA and parC associated with quinolone resistance were found in all phenotypically resistant isolates to ciprofloxacin and moxifloxacin (n = 14). Sixty-eight virulence genes were identified among isolates. Both lukD/E and lukM/F-PV-P83 were identified in 22.5% of isolates, all were non-ST398. Conclusion: In this study, ST398 had the highest potential to cause disease and had a massive prevalence in bovine mastitis cases. Five different sequence types and seven clusters were identified in the federal state of Thuringia. The circulation of some clusters in the same region over several years shows the persistence of cluster-associated infection despite the intensive veterinary care. On the other hand, some regions had different clusters at the same year or in different consecutive years. Different sequence types and associated different clusters of S. aureus were geographically widely distributed among dairy farms in Thuringia. The findings of this study show that various clusters have the potential to spread over a large geographical scale. The detection of LA-MRSA on dairy farms, which is known for cabapility to widely spread among different groups of animals, humans and their environment urges for the implementation of national wide strategic programs. The identification of CA-MRSA among the isolates such as ST398 poses a significant risk for the transmission of such strains between animals and humans on dairy farms.

Genomic insight into Campylobacter jejuni isolated from commercial turkey flocks in Germany using whole-genome sequencing analysis.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0-26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain-Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (bla OXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3')-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 ß-lactam resistance genes (bla OXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Detection and Phylogenetic Analysis of Extended-Spectrum ß-Lactamase (ESBL)-Genetic Determinants in Gram-Negative Fecal-Microbiota of Wild Birds and Chicken Originated at Trimmu Barrage.

Extended-spectrum ß-lactamases (ESBL) give rise to resistance against penicillin and cephalosporin antibiotics in multiple bacterial species. The present study was conducted to map genetic determinants and related attributes of ESBL-producing bacteria in three wild aquatic bird species and chickens at the "Trimmu Barrage" in district Jhang, Punjab province, Pakistan. To study the prevalence of ESBL-producing bacteria, a total of 280 representative samples were collected from wild bird species; cattle egrets (Bubulcus ibis), little egrets (Egretta garzetta) and common teals (Anas crecca) as well as from indigenous chickens (Gallus gallus domesticus) originating from a local wet market. The isolates were confirmed as ESBL producers using a double disc synergy test (DDST) and bacterial species were identified using API-20E and 20NE strips. A polymerase chain reaction (PCR) was used to detect ESBL genetic determinants and for genus identification via 16S rRNA gene amplification. A phenotypic antimicrobial susceptibility test was performed for ESBL-producing isolates against 12 clinically relevant antibiotics using the Kirby-Bauer disk diffusion susceptibility test. A phylogenetic tree was constructed for the sequence data obtained in this study and comparative sequence data obtained from GenBank. The overall prevalence of ESBL-producing bacteria was 34.64% (97/280). The highest percentage (44.28%; 31/70) of ESBL-producing bacteria was recovered from chickens (Gallus gallus domesticus), followed by little egrets (Egretta garzetta) (41.43%; 29/70), common teal (Anas crecca) (28.57%; 20/70) and cattle egrets (Bubulcus ibis) (24.28%; 17/70). Five different ESBL-producing bacteria were identified biochemically and confirmed via 16S rRNA gene sequencing, which included Escherichia coli (72; 74.23%), Enterobacter cloacae (11; 11.34%), Klebsiella pneumoniae (8; 8.25%), Salmonella enterica (4; 4.12%) and Pseudomonas aeruginosa (2; 2.06%). Based on PCR, the frequency of obtained ESBL genes in 97 isolates was blaCTX-M (51.55%), blaTEM (20.62%), blaOXA (6.18%) and blaSHV (2.06%). In addition, gene combinations blaCTX-M + blaTEM, blaTEM + blaOXA and blaCTX-M + blaSHV were also detected in 16.49%, 2.06% and 1.03% of isolates, respectively. The ESBL gene variation was significant (p = 0.02) in different bacterial species while non-significant in relation to different bird species (p = 0.85). Phylogenetic analysis of amino acid sequence data confirmed the existence of CTX-M-15 and TEM betalactamases. The average susceptibility of the antibiotics panel used was lowest for both Klebsiella pneumoniae (62.5% ± 24.42) and Salmonella enterica (62.5% ± 31.08) as compared to Enterobacter cloacae (65.90% ± 21.62), Pseudomonas aeruginosa (70.83% ± 33.42) and Escherichia coli (73.83% ± 26.19). This study provides insight into the role of aquatic wild birds as reservoirs of ESBL-producing bacteria at Trimmu Barrage, Punjab, Pakistan. Hence, active bio-surveillance and environment preservation actions are necessitated to curb antimicrobial resistance.

Cross-Sectional Study for Detection and Risk Factor Analysis of ESBL-Producing Avian Pathogenic Escherichia coli Associated with Backyard Chickens in Pakistan.

The increasing incidence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia (E.) coli in backyard chicken farming in Pakistan is of serious concern. This study aimed to assess the prevalence, antimicrobial resistance patterns and risk factors associated with ESBL avian pathogenic E. coli (APEC) isolated from backyard chickens in the Jhang district, Punjab, Pakistan. In total, 320 cloacal swabs were collected from four breeds of backyard chicken (Aseel, Golden, Misri and Necked Neck). ESBL E. coli were phenotypically identified using double disc synergy test (DDST) and corresponding genes were confirmed by multiplex polymerase chain reaction (mPCR). Out of the 320 samples, 164 (51.3%) were confirmed as E. coli, while 74 (45.1%) were characterized as ESBL E. coli. The frequency of isolation of ESBL E. coli was highest in Aseel chickens (35.1%). Of the 164 confirmed E. coli, 95.1%, 78.6%, 76.8%, 71.3%, 70.1%, 68.9%, 60.4% and 57.3% were resistant against tylosin, doxycycline, cefotaxime, enrofloxacin, colistin, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin, respectively. The ESBL gene types detected and their corresponding proportions were blaCTX-M (54.1 %, 40/74), blaTEM, (12.2%, 9/74) and co-existence (blaCTX-M and blaTEM) were shown in 33.8% (25/74). The blaCTX-M gene sequence showed homology to blaCTX-M-15 from clinical isolates. The mean multiple antibiotic resistance index (MARI) was found to be higher among ESBL E. coli (0.25) when compared to non-ESBL E. coli (0.17). Both free-range husbandry management system (p = 0.02, OR: 30.00, 95% CI = 1.47-611.79) and high antimicrobial usage in the last 6 months (p = 0.01, OR: 25.17, 95% CI = 1.81-348.71) were found significantly associated with isolation of ESBL-producing E. coli in the tested samples using binary logistic regression analysis. This study confirmed the potential of backyard chickens as a reservoir for ESBL E. coli in the Jhang district, Punjab, Pakistan.

Zoonotic agents in small ruminants kept on city farms in southern Germany.

Sheep and goats are popular examples of livestock kept on city farms. In these settings, close contacts between humans and animals frequently occur. Although it is widely accepted that small ruminants can carry numerous zoonotic agents, it is unknown which of these agents actually occur in sheep and goats on city farms in Germany. We sampled feces and nasal liquid of 48 animals (28 goats, 20 sheep) distributed in 7 city farms and on one activity playground in southern Germany. We found that 100% of the sampled sheep and 89.3% of the goats carried Shiga toxin-producing Escherichia coli (STEC). The presence of Staphylococcus spp. in 75% of both sheep and goats could be demonstrated. Campylobacter spp. were detected in 25% and 14.3% of the sheep and goats, respectively. Neither Salmonella spp. nor Coxiella burnetii was found. On the basis of these data, we propose a reasonable hygiene scheme to prevent transmission of zoonotic agents during city farm visits.

Determination of antimicrobial sensitivities of Campylobacter jejuni isolated from commercial turkey farms in Germany.

The emergence of antimicrobial resistance among Campylobacter isolates recovered from turkeys has increased dramatically. Monitoring the progress of this resistance becomes a growing public health issue. The aim of the present study was to provide information of the current status of antibiotic resistance patterns in Campylobacter jejuni from turkeys. Seventy-six C. jejuni isolates were recovered from 67 epidemiologically unrelated meat turkey flocks in different regions of Germany in 2010 and 2011. The isolates were typed by flaA genotyping and were investigated for antimicrobial susceptibility against 12 antibiotics by using a broth microdilution test as well as testing the genetic determination of ciprofloxacin, tetracycline, and erythromycin resistance. All isolates (n = 76) were sensitive to gentamicin and chloramphenicol. The numbers of isolates that were sensitive to streptomycin, erythromycin, neomycin, and amoxicillin were 69 (90.8%), 61 (80.2%), 58 (76.4%), and 44 (57.9%), respectively. Only one isolate was sensitive to all tested antibiotics. The emergence of a high resistance rate and multidrug resistance to three or more classes of antimicrobial agents were observed. The resistance against sulphamethoxazole/trimethoprim, metronidazole, ciprofloxacin, naladixic acid, and tetracycline was 58 (76.3%), 58 (76.3%), 53 (69.7%), 51 (67.1%), and 42 (55.3%), respectively. None of the isolates was resistant to all antibiotics. Multidrug resistance to three or more classes of antimicrobial agents was found and ranged from 3.9% to 40.8%. Replacement of the Thr-86-->Ile in gyrA gene and detection of the tet(O) gene were the main resistance mechanisms for fluoroquinolones and tetracycline, respectively, while the lack of mutation in position 2074 and 2075 on the 23S rRNA gene was responsible for macrolide resistance. The phenotypic and genotypic resistance profiles were compatible in the case of ciprofloxacin and tetracycline but were not completely congruent with respect to erythromycin.

Serological Prevalence of and Risk Factors for Coxiella burnetti Infection in Women of Punjab Province, Pakistan.

Background: Coxiella burnetii, the etiological agent of Q (query) fever, provokes abortions in ruminants and is suspected to cause adverse pregnancy outcomes in women. Infection of pregnant women is linked with high mortality and morbidity of the fetus and the mother is at high risk to acquire chronic Q fever. This research was conducted to evaluate the prevalence of Q fever in women and to detect associated risk factors in four districts of Punjab Province, Pakistan. Methods: A total of 297 blood samples were obtained from 147 pregnant and 150 non-pregnant women of the districts Okara, Jhang, Chiniot and Faisalabad of Punjab, Pakistan. Data related to risk factors and demographic parameters were collected using a questionnaire. Serum samples were screened for phase I and phase II specific IgG antibodies for antigens of phase I and phase II using ELISA tests. Univariate and binary regression were used to analyze important risk factors of Q fever. Results: Twenty-five serum samples (8.4%) were found seropositive for Q fever. Seventeen women were positive for Phase-I and twenty-one were positive for phase-II antibodies. Highest and statistically significant (p < 0.05) seroprevalence of 17.1% was observed in Faisalabad. Age, urbanicity, living status, pregnancy status, abortion history, occupation, and consumption of tap water were positively correlated (p < 0.05) with Q fever, while being aged, urbanity, low income, contact with animals and consumption of tap water was identified as potential risk factors. Conclusions: Q fever is prevalent in women of Pakistan. There is a need for an awareness program about the importance of C. burnetii infections and prevention strategies in women during pregnancy to minimize adverse pregnancy outcomes.