ORIGINAL ARTICLE

Environmental cephalosporin-resistant gram-negative bacteria: what is lurking beyond the hospital doors?

Yasna Yusuf¹, Olubosede Awoyomi², Alison Bradbury², Yongsheng Wang², Briana Episcopia², Marie Abdallah¹ and John Quale^1*

¹Division of Infectious Diseases, NYC H+H/Kings County, New York City, USA; ²Department of Infection Prevention and Control, NYC H+H/Kings County, New York City, USA

Abstract

Antibiotic resistant Gram-negative bacilli are being increasingly recognized in community-onset infections. Concerns have been raised regarding environmental surfaces as reservoirs for multidrug-resistant pathogens; in particular, community wastewater samples have been found to harbor cephalosporin- and carbapenem-resistant Enterobacterales. In this report, we used selective media to culture cephalosporin-resistant Gram-negative bacilli from high-touch surfaces in the environment surrounding hospitals in New York City. Of the 336 surfaces swabbed, 66 grew cephalosporin-resistant Gram-negative bacteria. No Enterobacterales were isolated. The most common isolate was Brucella (Ochrobactrum) spp., accounting for 26 of the 66 positive cultures; 19 originated from surfaces near two hospitals. One isolate of Brucella (Ochrobactrum) spp. was found to possess bla_OXA-24. Acinetobacter spp. accounted for 12 of the 66 positive cultures. Forty cultures obtained with saline-saturated gauze were obtained at public transportation centers near hospitals; a comparable 40 cultures were obtained at transportation centers distant from hospitals. Of the 38 positive cultures from transportation surfaces near medical centers, 15 grew Brucella (Ochrobactrum) spp. In comparison, Brucella (Ochrobactrum) spp. accounted for only five of the 33 positive transportation cultures distant from medical centers (P = 0.03). Again, none of the cultures grew Enterobacterales. It is reassuring that dried environmental surfaces around hospitals did not contain resistant Enterobacterales. However, Brucella (Ochrobactrum) spp. was widespread and territorial and may be a reservoir for OXA-type carbapenemases.

Keywords: environmental contamination; Brucella; Ochrobactrum; Acinetobacter; carbapenem resistance

 

 

Infections with multidrug-resistant Gram-negative bacteria are associated with poor outcomes and increased expenditures. In 2019, the Centers for Disease Control and Prevention identified carbapenem-resistant A. baumannii and Enterobacterales as ‘urgent threats’ and extended spectrum beta-lactamase (ESBL)-possessing Enterobacterales and multidrug-resistant P. aeruginosa as ‘serious threats’ (1).

Community-onset infections with ESBL Enterobacterales and carbapenem resistant bacteria are being increasingly recognized. ESBL-Enterobacterales have been recovered in water samples and animals; poor sanitation and overcrowding conditions contribute to community-onset infections with these pathogens (2, 3). Community onset cases of carbapenem-resistant Enterobacterales are also recognized. The finding of carbapenemase-producing Enterobacterales in water samples, including wastewater near hospitals, raises concern for community acquisition via environmental contamination (4–6).

Environmental contamination with multidrug-resistant non-fermenting Gram-negative pathogens has also been well documented. In particular, Acinetobacter spp. is well equipped to survive prolonged periods in arid conditions (7–9), and outbreaks of infections can follow natural disasters and conflicts (10). Similarly, both Pseudomonas spp. and Stenotrophomonas maltophilia can survive extended intervals in desiccated environments and are important multidrug-resistant pathogens (11, 12).

In this report, we searched for the presence of cephalosporin-resistant Gram-negative bacilli on environmental surfaces surrounding acute care medical centers in New York City.

Materials and methods

Two datasets of environmental cultures were gathered during July and August 2023. The first dataset involved swabbing high-touch environmental surfaces in areas near (generally within a two-block distance) of eight major medical centers located in the boroughs of Brooklyn, Manhattan, and Queens of New York City. Most cultures were from outside locations; a few cultures from inside the hospital lobby were permitted. Forty-two sterile rayon-tipped swabs (Puritan Medical Products, Guilford, ME) were first saturated in sterile saline. The environmental surface culture was obtained and inoculated into ~ 2 mLs of MacConkey broth containing 4 μg/mL of ceftazidime and 4 μg/mL of amphotericin B. A 43^rd swab served as a sterility control.

The second dataset involved environmental surfaces involving the eight public transportation centers nearby the medical centers; in addition, comparative environmental cultures from eight transportation centers (located in Brooklyn, Long Island, and New Jersey) distant from acute care medical centers were gathered. Five sterile gauze 2X2 inch pads were first saturated with normal saline. High touch areas outside of the transportation center were wiped with the gauze, and the gauze placed in ~ 20 mL of MacConkey broth containing ceftazidime and amphotericin B.

All cultures were incubated at 37°C for up to 48 h. All samples with suspected growth were subcultured on MacConkey agar. Individual colonies were isolated, and susceptibility testing involving ceftazidime and meropenem was performed by the broth microdilution method using cation-supplemented Mueller-Hinton broth (13); P. aeruginosa ATCC 27853 was used as a control. Isolates confirmed with ceftazidime MICs ≥ 8 μg/mL (non-susceptible for Enterobacterales by CLSI definitions) were identified by sequencing a fragment of the 16S rRNA gene using previously described primers and PCR conditions (14). Isolates were considered resistant to meropenem if the MIC was ≥ 4 μg/mL. The presence of the β-lactamases TEM, SHV, CTX-M, KPC, NDM, VIM, IMP, and OXA-type carbapenemases was screened using previously described PCR primers and conditions (15).

The survivability of different bacterial isolates in a desiccated environment was also examined. Bacterial isolates were grown to the late-log phase of growth in Mueller-Hinton broth. Cultures were vortexed, and 20 μL aliquots was placed in a sterile 96-well plate. The samples were allowed to dry, and the pellet was resuspended in 100 μL sterile saline at T = 0 (baseline), 3, and 7 days. Serial 1:10 dilutions of the suspension were subcultured on Mueller-Hinton agar plates and incubated overnight; the number of surviving bacteria in the pellet was then determined.

Results

Cultures from environmental surfaces near hospitals

There were 66 cephalosporin-resistant Gram-negative organisms identified from the 336 swab cultures from environmental surfaces near medical centers (Table 1). Among the 12 Acinetobacter spp., 16S sequencing identified four isolates most consistent with A. calcoaceticus/A. soli/A. seifertei and four isolates consistent with A. ursingii/A. septicum; one isolate was identified as A. baumannii. Five of the Acinetobacter spp., including the A. baumannii, were identified in hospital lobby surfaces. All three of the isolates of Pseudomonas spp. came from environmental surfaces near a single hospital; two were most closely identified as P. putida/fulva species. One culture, from a mailbox handle, yielded Stenotrophomonas maltophilia. The most common organism isolated from the environment was Brucella (Ochrobactrum) spp. Of the 26 isolates, 15 were identified most closely with B. intermedia/haematophila and eight at B. anthropi. One isolate of B. intermedia/haematophila, from the surface of a vending machine, was found to possess the carbapenemase OXA-24. Outdoor gate and door handles, as well as handrails, were common sites positive for Brucella (Ochrobactrum) spp. Although seven of the eight hospitals had Brucella (Ochrobactrum) spp. in their environment, two hospitals accounted for 19 of the 26 cultures. Not one member of the Enterobacterales order was recovered in the environmental cultures.

Table 1. Ceftazidime-resistant Gram-negative bacilli recovered in environmental swab samples near major medical centers in the New York City

Isolate	Number (% resistant to meropenem)
Achromobacter spp.	3 (33)
Acinetobacter spp.	12 (8)
Brevundimonas spp.	7 (57)
Brucella (Ochrobactrum) spp.	26 (4)
Cupriavidus spp.	5 (100)
Pseudomonas spp.	3 (33)
Rhizobium/Agrobacterium spp.	6 (17)
Stenotrophomonas spp.	1 (100)
Other*	3 (33)
*Halotalea spp. (n = 1) and Pantoea spp. (n = 2).

Cultures from transportation centers near and far medical centers

Among the 40 cultures taken at public transportation centers (i.e. subway stations and bus stops) near hospitals, 38 bacteria were recovered (Table 2). Among eight Acinetobacter isolates, 16S sequencing identified three isolates most consistent with A. calcoaceticus/A. soli/A. seifertei and two isolates consistent with A. ursingii/A. septicum. No isolates of Pseudomonas were recovered, and two isolates of carbapenem-resistant Stenotrophomonas were isolated. Fifteen of the 38 isolates were Brucella (Ochrobactrum) spp., including 14 B. intermedia/haematophila and one isolate of B. anthropi.

Table 2. Comparison of environmental cultures taken at transportation centers near and distant from medical centers

Isolate	Samples near medical centers	Samples far from medical centers
Number (% carbapenem resistant)
Achromobacter spp.	2 (0)	2 (0)
Acinetobacter spp.	8 (0)	6 (0)
Brevundimonas spp.	4 (25)	3 (0)
Brucella (Ochrobactrum) spp.	15 (20)	5* (20)
Cupriavidus spp.	6 (100)	8 (100)
Pseudomonas spp.	0	2 (50)
Rhizobium/Agrobacterium spp.	1 (0)	2 (0)
Stenotrophomonas spp.	2 (100)	5 (100)
* P = 0.03 compared to samples near medical centers.

A total of 33 isolates were recovered among the 40 cultures taken at transportation centers distant from medical centers. Among six Acinetobacter spp., two were identified as A. baumannii. There were two Pseudomonas spp. isolates and five isolates of carbapenem-resistant S. maltophilia. Five isolates were Brucella (Ochrobactrum) spp., with four identified as B. pseudogrignonensis. Compared to the cultures obtained near medical centers, there were significantly fewer isolates of Brucella (Ochrobactrum) spp. from cultures obtained far from medical centers (5 of 33 vs. 15 of 38, P = 0.03). Once again, no Enterobacterales were isolated in the environmental samples.

Comparison of the survivability of bacteria in desiccated conditions

The survival rates of six bacteria under desiccated conditions were determined. Survival of E coli ATCC 25922 and K pneumoniae ATCC 700603 was compared to four isolates gathered from the environmental samples: two isolates of B. intermedia/haematophila and one isolate each of P. putida/P. fulva and S. maltophilia (Table 3). With a starting inoculum of ~ 10⁶ CFU, survival was considerably poorer for the two members of Enterobacterales (and especially E. coli). Over the course of 7 days, there was only a 1–2 log10 CFU decrease in the starting inoculum for the isolates of Brucella (Ochrobactrum) spp. and S. maltophilia.

Table 3. Decrease in log10 CFU of bacteria over 3 and 7 days at room temperature under desiccant conditions

	E. coli	K. pneumoniae	B. intermedia/haematophila	P putida/fulva	B. intermedia/haematophila	S. maltophilia
Day 3	6.15	2.06	1.06	1.35	1.00	0.15
Day 7	6.15	3.18	1.76	4.73	1.10	1.79

Discussion

It was reassuring that cephalosporin-resistant Enterobacterales were not recovered from 416 peri-hospital environmental surfaces in our metropolitan region. The routine use of hand hygiene products by hospital employees when leaving the patient care areas may contribute to these findings. Reports of antibiotic-resistant Enterobacterales in the environment have generally emphasized water-related samples, including sink basins, rivers, and wastewater areas (2, 4, 5, 16, 17). Our samples involved ‘high-touch’ dry surfaces only. As noted in this report, compared to Enterobacterales spp., certain non-fermentative Gram-negative bacteria have a distinct survival advantage in arid environments, likely accounting for our findings. While encouraging that most dry surfaces outside of hospitals are not heavily contaminated with ESBL- or carbapenemase-producing Enterobacterales, testing of standing water and hospital wastewater samples should be done to further evaluate the environmental ecology of these areas.

All of our positive cultures yielded non-fermenting Gram-negative bacilli. Of the 139 positive environmental cultures in this study, 26 (19%) belonged to the genus Acinetobacter, including many to the A. calcoaceticus – A. baumannii complex. The A. calcoaceticus – Acinetobacter baumannii complex, and in particular A. baumannii, has been shown to survive quite well on environmental surfaces within hospitals (7–9). Our cultures were taken during the summer months of July and August in New York City; the high rate of contamination we observed suggests the extra-hospital environment may serve as an important reservoir for hospital-associated Acinetobacter infections, especially during the summer months when there is an increase in Acinetobacter-related infections (18). Similarly, S. maltophilia is another important multidrug-resistant hospital pathogen that can survive prolonged periods on desiccated surfaces (12). The finding that 6% of our positive cultures, especially those involving transportation areas, also suggest the extra-hospital environment may be an important reservoir for introduction into the hospital setting. Whole genome sequencing of extra-hospital and clinical isolates of A. baumannii and S. trophomonas would determine if these isolates are related.

Perhaps our most unanticipated finding was the widespread recovery of Brucella (Ochrobactrum) spp. The reclassification of Ochrobactrum spp. into the Brucella genus in 2022 was followed by considerable controversy (19, 20). Classic Brucella spp. (including B. abortus, B. canis, B. melitensis, and B. suis) are reportable pathogens that cause animal and human infections (19–21). The reclassified organisms, designated Brucella (Ochrobactrum) spp., are not reportable, but infections in humans are well described (19–21).

Brucella (Ochrobactrum) spp. is well equipped for survival under harsh conditions. Survival in the environment is enhanced by heavy metal tolerance and degradation of xenobiotic compounds including toluene, hexane, and p-xylene (22, 23). In our study, Brucella (Ochrobactrum) spp. accounted for 39% of the cephalosporin-resistant bacteria isolated in the extra-hospital surfaces and appeared to be territorial. In addition, the proportion of Brucella (Ochrobactrum) spp. in the transportation centers near the hospitals was significantly greater than that for centers distant from hospitals. Further study will be needed to determine the selective pressures (e.g. environmental cleaning protocols and use of hospital hand hygiene products) responsible for this predominance. Again, whole genome sequencing would be helpful in determining if a single strain is involved in this widespread environmental contamination.

Brucella (Ochrobactrum) spp. possesses intrinsic resistance to tetracycline, erythromycin, novobiocin, and because of an AmpC β-lactamase, penicillins, and cephalosporins (22–24). Other resistance genes, including those involving polymyxin and fluoroquinolones, have also been documented (22). Recommended therapies for infections due to Brucella (Ochrobactrum) spp. include fluoroquinolones, co-trimazole, and imipenem (19, 23, 24). The finding of OXA-24 β-lactamase, a carbapenemase typically found in Acinetobacter spp. (25, 26), in one of our isolates suggests that these bacteria can also be reservoirs of clinically important antibiotic resistance genes.

While our study provides reassurance that cephalosporin-resistant Enterobacterales are not widespread on extra-hospital surfaces, we did confirm the presence of an unexpected number of Acinetobacter spp. In addition, a surprising number of Brucella (Ochrobactrum) spp. were recovered, including one harboring an OXA carbapenemase.

Acknowledgments

None.

Ethics approval

Declared exempt by the SUNY Downstate Institutional Review Board.

References

1.	CDC. Antibiotic resistance threats in the United States, 2019. Atlanta, GA: U.S. Department of Health and Human Services, CDC; 2019.
2.	Cocker D, Chidziwisano K, Mphasa M, Mwapasa T, Lewis JM, Rowlingson B, et al. Investigating One Health risks for human colonisation with extended spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Malawian households: a longitudinal cohort study. Lancet Microbe 2023; 4: e534–43. doi: 10.1016/S2666-5247(23)00062-9
3.	Paumier A, Asquier-Khati A, Thibaut S, Coeffic T, Lemenand O, Larramendy S, et al. Assessment of factors associated with community-acquired extended-spectrum β-lactamase-producing Escherichia coli urinary tract infections in France. JAMA Netw Open 2022; 5: e2232679. doi: 10.1001/jamanetworkopen.2022.32679
4.	Lee J, Sunny S, Nazarian E, Fornek M, Abdallah M, Episcopia B, et al. Carbapenem-resistant Klebsiella pneumoniae in large public acute-care healthcare system, New York, New York, USA, 2016–2022. Emerg Infect Dis 2023; 29: 1973–8. doi: 10.3201/eid2910.230153
5.	Islam MA, Islam M, Hasan R, Hossain MI, Nabi A, Rahman M, et al. Environmental spread of New Delhi metallo-β-Lactamase-1-producing multidrug-resistant bacteria in Dhaka, Bangladesh. Appl Environ Microbiol 2017; 83: e00793-17. doi: 10.1128/AEM.00793-17
6.	Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 2011; 11: 355–62. doi: 10.1016/S1473-3099(11)70059-7
7.	Jawad A, Seifert H, Snelling AM, Heritage J, Hawkey PM. Survival of Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates. J Clin Microbiol 1998; 36: 1938–41. doi: 10.1128/JCM.36.7.1938-1941
8.	Fahy S, O’Connor JA, Lucey B, Sleator RD. Hospital reservoirs of multidrug resistant Acinetobacter species-The elephant in the room! Br J Biomed Sci 2023; 80: 11098. doi: 10.3389/bjbs.2023.11098
9.	Antunes LC, Visca P, Towner KJ. Acinetobacter baumannii: evolution of a global pathogen. Pathog Dis 2014; 71: 292–301. doi: 10.1111/2049-632X.12125
10.	Joly-Guillou, M-L. Clinical impact and pathogenicity of Acinetobacter. Clin Microbiol Infect 2005; 11: 868–73. doi: 10.1111/j.1469-0691.2005.01227
11.	Saati-Santamaría Z, Baroncelli R, Rivas R, García-Fraile P. Comparative genomics of the genus Pseudomonas reveals host- and environment-specific evolution. Microbiol Spectr 2022; 10: e0237022. doi: 10.1128/spectrum.02370-22
12.	Brooke JS. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 2012; 25: 2–41. doi: 10.1128/cmr.00019-11
13.	Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 28th informational supplement, M100-S33. Wayne, PA: CLSI; 2023.
14.	Srinivasan R, Karaoz U, Volegova M, MacKichan J, Kato-Maeda M, Miller M, et al. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS One 2015; 10: e0117617. doi: 10.1371/journal.pone.0117617
15.	Asrat H, Samaroo-Campbell J, Ata S, Quale J. Contribution of iron-transport systems and β-lactamases to cefiderocol resistance in clinical isolates of Acinetobacter baumannii endemic to New York City. Antimicrob Agents Chemother 2023; 67: e0023423. doi: 10.1128/aac.00234-23
16.	Muzslay M, Moore G, Alhussaini N, Wilson APR. ESBL-producing Gram-negative organisms in the healthcare environment as a source of genetic material for resistance in human infections. J Hosp Infect 2017; 95: 59–64. doi: 10.1016/j.jhin.2016.09.009
17.	Mills MC, Lee J. The threat of carbapenem-resistant bacteria in the environment: evidence of widespread contamination of reservoirs at a global scale. Environ Pollut 2019; 255: 113143. doi: 10.1016/j.envpol.2019.113143
18.	McDonald LC, Banerjee SN, Jarvis WR. Seasonal variation of acinetobacter infections: 1987–1996. Nosocomial Infections Surveillance System. Clin Infect Dis 1999; 29: 1133–7. doi: 10.1086/313441
19.	Moreno E, Blasco JM, Letesson JJ, Gorvel JP, Moriyón I. Pathogenicity and its implications in taxonomy: the Brucella and Ochrobactrum case. Pathogens 2022; 11: 377. doi: 10.3390/pathogens11030377
20.	Moreno E, Middlebrook EA, Altamirano-Silva P, Al Dahouk S, Araj GF, Arce-Gorvel V, et al. If you’re not confused, you’re not paying attention: Ochrobactrum is not Brucella. J Clin Microbiol 2023; 61: e0043823. doi: 10.1128/jcm.00438-23
21.	American Society for Microbiology. Brucella and Ochrobactrum taxonomic updates for laboratories. Available from: ASM.org [cited 15 February 2024].
22.	Gohil K, Rajput V, Dharne M. Pan-genomics of Ochrobactrum species from clinical and environmental origins reveals distinct populations and possible links. Genomics 2020; 112: 3003–12. doi: 10.1016/j.ygeno.2020.04.030
23.	Ryan MP, Pembroke JT. The genus Ochrobactrum as major opportunistic pathogens. Microorganisms 2020; 8: 1797. doi: 10.3390/microorganisms8111797
24.	Thoma B, Straube E, Scholz HC, Dahouk SA, Zöller L, Pfeffer M, et al. Identification and antimicrobial susceptibilities of Ochrobactrum spp. Int J Med Microbiol 2009; 299: 209–20. doi: 10.1016/j.ijmm.2008.06.009
25.	Bou G, Oliver A, Martínez-Beltrán J. OXA-24, a novel class D beta-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain. Antimicrob Agents Chemother 2000; 44: 1556–61. doi: 10.1128/AAC.44.6.1556-1561.2000
26.	Lasarte-Monterrubio C, Guijarro-Sánchez P, Bellés A, Vázquez-Ucha JC, Arca-Suárez J, Fernández-Lozano C, et al. Carbapenem resistance in Acinetobacter nosocomialis and Acinetobacter junii conferred by acquisition of blaOXA-24/40 and genetic characterization of the transmission mechanism between Acinetobacter genomic species. Microbiol Spectr 2022; 10: e0273421. doi: 10.1128/spectrum.02734-21